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EXPRESSION SYSTEMS
EXTRACTION AND CLARIFICATION
PURIFICATION
CHARACTERIZATION
OTHERS

 
PURIFICATION PROTOCOLS
Dr. Mario Lebendiker
mariol@cc.huji.ac.il
Tel: 972-2-6586920

mario



Purification Strategy - and more

Test Tube

Removal of Nucleic Acids Buffer for Tag Purif

Cleaning and Regeneration of Resins

Columns

High-throughput process development (HTPD)

Purification of large biomolecules

Purification of Recombinant Proteins

GST
poly His
Maltose
Calmodulin
Intein
 T7.Tag
Cellul.Bind.Domain
Nus A
Biotinylated 
SUMO
StrepTag
FLAG
Halo tag Fusion
Solubility enhancement tags (SETs)
Fluorescent Protein
Cold Expression
LYTAG Two PhaseSystem
Profinity eXact™
Bispecific Affinity
CaptureSelect C‐tag

Cleavage of Recombinant Proteins

Cleavage Sites Table for Fusion Proteins

Factor-Xa
Thrombin
Enterokinase
PreScission
TAGZyme
TEV-Protease
HRV 3C
SUMO

Affinity Chromatography
Activated resins
Antibody Purification and Ab. fragmentation



Gel Filtration

Ion Exchange Chromatography
Multimodal and Hydrophobic Charge-Induction Chromatography


Thiophilic Chromatography

Reverse Phase Chromatography

Expanded Bed Adsorption

Phospho-Protein Purification

Hydroxyapatite

Protein Labeling with Biotin

 Polyubiquitin-modified proteins

Deglycosylation

Purification of DNA Binding Proteins

Viral Purification


High Abundant Serum Proteins Removal
Protein Refolding / Inclusion Bodies

Storage of Purified Proteins

Protein Aggregation

Crystallography and Recombinant Methods

Simulated Moving Bed Chromatography

Purification Strategy - Others 

       GE-HEALTHCARE  Handbook Protein Purification (pdf)  
       GE-HEALTHCARE  Strategies for Protein Purif ication Handbook  (pdf)  
       GE-HEALTHCARE  Protein purification Applications  (pdf)      
       GE-HEALTHCARE  Protein and Peptide Purification - Technique selection guide  (pdf)
       GE-HEALTHCARE  Bio Process - Product guide   (pdf)
       GE-HEALTHCARE  Purifying Challenging Proteins: Membrane proteins, Multiprotein complexes and Inclusion bodies  (pdf)
       GE-HEALTHCARE  ÄKTAdesign Purification Method handbook. This handbook is intended to help you make full use of your ÄKTAexplorer™
            or ÄKTApurifier™ chromatography system.The handbook is a collection of useful step-by-step protocols to aid your everyday purification work.
            Detailed instructions and recommendations are presented in a straightforward format, which should be easy to follow without needing a high level
            of expertise in programming or in chromatography.
 (pdf)
      GE-HEALTHCARE  Purification of tagged proteins   (pdf)
      GE-HEALTHCARE  Packing of Gel Filtration column  (movie)
      GE-HEALTHCARE  Packing of IEX, Affinity or HIC column  (movie)
      GE-HEALTHCARE  Column evaluation (movie)  
      GE-HEALTHCARE  PreDictor plates for High-throughput process development (HTPD). Disposable 96-well filter plates prefilled with GE Healthcare
            BioProcess™     chromatography media (Capto™ Q, S, DEAE, MMC, Adhere; Q and SP Sepharose™; MabSelect™, SuRe™ and Xtra™;
            AIEX and CIEX screening plate. Support HTPD by allowing parallel screening of chromatographic conditions. They can be used in automated
            workflows using robotic systems, or can be operated  manually using multi-channel pipettes   (pdf)   (pdf Handbook)
        BIA Separations  CIM® Monolithic Columns  based on CIM Convective Interaction Media® Technology; suitable for purification of large biomolecules
           such as viruses (viral vectors and vaccines), DNA (plasmid DNA) and larger proteins (Immunoglobulins G and M, pegylated proteins). CIM® Monolithic
           Columns exhibit unrivaled characteristics in terms of operational flow rates, binding capacity and separation resolution for large biomolecules. Products
            are used in research, laboratory, pilot and  industrial production stages and are extremely simple to use, with no packing of columns needed
            (pdf-I)  (pdf-II)  (pdf-III)  
        BIO RAD Sample Preparation Handbook   (pdf)
        CLONTECH:  Troubleshooting Guide   (pdf)
        MERCK    Cleaning and Regeneraton of  Fractogel EMD sorbents   (pdf)
        NOVAGEN   Benzonase Nuclease - Removal of Nucleic Acids(pdf)
        NOVAGENBuffer Compositin for Fusion Tag Affinity Purification (pdf)  
       
Pall    Chromatography Media Selection Guide   (pdf)              
            Protein Sample Preparation and Analysis Application Manual. Table of contents   (pdf)
               1.0 Introduction:
1.1 Proteomics Process Flo -. 1.2 Product Selection  (pdf1)  
               2.0 Pre-Analytical Applications:
2.1 Abundant Protein Removal - 2.2 Protein Fractionation - 2.3 Detergent Removal - 2.4 Concentration, Desalting,
            and Buffer Exchange - 2.5 Generic Clarification of Samples by Microporous Filtration (Particulate Removal) - 2.6 Endotoxin Removal  (pdf2)
                3.0 Analysis:
3.1 Western Blotting - 3.2 Affinity Activated or Activatable Membranes  (pdf3)
                4.0 Purification Applications: 4.1 Mustang® Ion Exchange Membranes - 4.2 BioSepra® Chromatography Resins - 4.3 Rapid Purification                                                     
           Development/Scouting (Process Proteomics) - 4.4 Buffer Exchange, Desalting, and Concentration - 4.5 Final Product Clarification  (pdf4)
                5.0 Supporting Technologies: 5.1 Water Supply - 5.2 Microbiology Testing   (pdf5)
                6.0 Technical Appendices:   6.1 Evaluation of Chromatography Column Packing Efficiency -  6.2 Optional Pre-Treatment to Improve Recovery
            of Samples from Ultrafiltration Spin Filters -  6.3 Vacuum Manifold for Use with Multi-Well Filter Plates -  6.4 General Filtration Procedures for
            AcroPrep™ and AcroWell™ Filter Plates  - 6.5 Chromatography Products Selection Guide - 6.6 BioSepra® Media - 6.7 Protein Sample Preparation
            and Analysis Media - 6.8 Chemical Compatibility Guide  (pdf6)
        
PIERCE:   Peptide solubility guidelines. Use amino acid characteristics to predict hydrophobicity.  (pdf)
         
TOSOH   Chromatographic Media Catalog   (pdf)
         TOSOH   TOYOPEARL® Orientation Sheet: Size Exclusion Chromatography, Ion Exchange Chromatography, Hydrophobic Interaction Chromatography  
              and Affinity Chromatography  (pdf)  (pdf-II)

        
TOSOH   General Principles of Chromatography. Poster   (pdf)  
         Removal of Nucleic Acids - Various Protocols
    
         Test Tube

       Columns   

       GE-HEALTHCARE    columns are designed for standard liquid chromatography of macromolecules. XK columns (pdf)  Tricorn  (pdf)  
                C-series columns  (pdf)   HR columns  (pdf)   HiScale™ columns (pdf)  
      GE-HEALTHCARE  Packing of Gel Filtration column  (movie)
      GE-HEALTHCARE  Packing of IEX, Affinity or HIC column  (movie)
      GE-HEALTHCARE  Column evaluation (movie)    
      Essential Life Solutions:   SNAP® Laboratory Glass Columns for high-performance preparative chromatography  (pdf-literature)  (pdf-tech manual)
                (video)  
       Pall    Pall® LRC Columns Chromatography Columns for Laboratory Applications  (pdf)   User Guide  (pdf-II) 


Purification of Recombinant Proteins 

      Structural Genomics Consortium  Protein production and purification. Nature Methods 2008, 5: 135-146  (pdf-I)  (pdf-II)
      GE-HEALTHCARE    The Recombinant Protein Handbook  (pdf-I)  (pdf-II)
      GE-HEALTHCARE  Purification of tagged proteins   (pdf)
      PIERCE:   Fusion Protein Fusion Protein Products   (pdf)
      SIGMA:   Detection and Purification Selection Guide  (pdf-I)    (pdf-II)

GST-Fusion Proteins

    Small scale GST-fusion protein purification under nature conditions
    
ABT :   Glutathione Agarose resins for use in affinity purification of Glutathione-S-transferase (GST) and GST tagged fusion proteins.  
        Bulk and Cartridges formats suitable for MPLC, FPLC™, ÄKTA™ design, batch, gravity, peristaltic pump & syringe
(pdf-I) (pdf-II)   (pdf-III)
        Troubleshooting  
(pdf-IV)
       CLONTECH: Glutathione Resins  (pdf)
       CLONTECH:  GST products Handbook (pdf)
       CLONTECH:  Protein Purification Products  (pdf)
       GE-HEALTHCARE  GST fusion protein Handbook   (pdf)
       GE-HEALTHCARE  Purification of tagged proteins   (pdf) 
       
GE-HEALTHCARE  Glutathione Sepharose™ High Performance is an affinity chromatography medium designed for easy, one-step
            purification of glutathione S-transferase (GST) tagged proteins
 (pdf)
       GE-HEALTHCARE  Separation of DnaK from pGEX-GST by ion exchange chromatography  (pdf)  
       
JENA    Glutathione ChroMatrix™, Fast Flow and GST Cleavage Capture Kit    (pdf)
       NOVAGENGST.Bind Kits(pdf) 
       NOVAGEN: His-Tag GST-Tag purification and Detection tools  (pdf)
       NOVAGEN:   Protein Purification and Detection. GST•Tag™ Fusions (pg.30-36)   (pdf)

       
poly His-Tagged Fusion Proteins 

     Small an large scale His-Tag fusion protein purification under nature conditions
      Small scale His-Tag fusion protein purification under denaturative conditions
      Protein Refolding on IMAC resin - Batch Screening Procedure - On-Column Scale-up
      ABT   offers affinity tag chelating resins for purifications of his-tag proteins by Immobilized Metal Affinity Chromatography (IMAC).
            Selection of metal chelating beads ( 4 different metals). Two different degrees of loading ( high and low density). Three different formats :bulk resins,
            pre-packed columns and cartridges.
(pdf)
            Procedure for using chelating agarose beads test kits (pdf-I) .  Troubleshooting  (pdf-II)
        ACTIVE MOTIVE: Ni-Ted Manual   (pdf)
        AFFILAND: Metal Purification  
        BioToolomics:  IMAC SepFast BG Media  (pdf-I)  Super Spin columns instructions  (pdf-II)
        CLONTECH:  BD TalonTM Metal Affinity Resins User Manual   (pdf)   (pdf-II) 
        CLONTECH: Talon Resin Protocol (pdf)    
        CLONTECH: Talon Products (pdf)   
        CLONTECHHisTALON™ Superflow™ 1 ml & 5 ml Cartridges User Manual  (pdf)
        CLONTECH:  TALON spin™ Columns Protocol  (pdf)
        CLONTECH:  Purification in the presence of Beta Mercaptoethanol  (pdf)     
        CLONTECH:  Protein Purification Products  (pdf)
        CLONTECH:  BD HAT Protein Expression and Purification System User Manual  (pdf)
        DYNAL  Dynabeads TalonTM  (pdf) 
        GE-HEALTHCARE  Purification of tagged proteins   (pdf)     
        GE-HEALTHCARE   Hi Trap Chelating (pdf)
        GE-HEALTHCARE   Hi Trap Chelating Application Note   (pdf)
        GE-HEALTHCARE   Chelating Sepharose Fast Flow Instructions   (pdf)
        GE-HEALTHCARE   His GraviTrap prepacked, single-use column for purification of histidine-tagged proteins by immobilized metal affinity chromatography
            (IMAC) without any need for a purification system. Negligible nickel leakage and is compatible with denaturing and
 reducing agents as well as a wide
            range of additives. Short purification times. High protein binding capacity, ≈ 40 mg/column. Purifies
unclarified samples  (pdf)     
       
GE-HEALTHCARE    His SpinTrap™ is a prepacked, single-use spin column for purifying histidine-tagged proteins by immobilized metal ion affinity
            chromatography (IMAC). The resin has high protein binding capacity, low nickel ion (Ni2+) leakage, and excellent compatibility  with denaturing agents
            plus a wide range of additives. His SpinTrap is used with a standard microcentrifuge and one purification run takes
approx. 10 min. His SpinTrap columns
            allow direct purification of unclarified cell lysates.
  (pdf-I)   (pdf-II)  
       
GE-HEALTHCARE   HisTrap™ FF crude. This pre-prepacked column is intended for preparative purification of  histidine-tagged recombinant proteins
            from unclarified lysate without precentrifugation and filtration of the sample. Leakage of Ni2+   is very low. The medium is compatible with all commonly
            used aqueous buffers, reducing agents, denaturants such as 6 M Gua-HCl
  and 8 M urea, and a range of other additives (pdf)     
       
GE-HEALTHCARE    Ni Sepharose and IMAC Sepharose selection guide  (pdf)         
       
GE-HEALTHCARE   IMAC Sepharose High Performance. Possible to charge with various metal ions for optimized selectivity. High protein binding
            capacity. Compatible with commonly used reducing agents, such as DTT, DTE, TCEP, and β-mercaptoethanol.
Stable in a wide range of denaturants,
            detergents, and buffer systems.
(pdf)  
       
GE-HEALTHCARE    Ni Sepharose High Performance  (pdf)   (pdf-II)     
       GE-HEALTHCARE    Article: Addition of imidazole during binding improves purity of histidine-tagged proteins  (pdf)
       GE-HEALTHCARE    Article: Snags with tags: Some observations made with (His)6-tagged proteins. The need to remove or omit the affinity tag from the
            purification scheme.P.Ramage et.al  (pdf)
       IBA  Tools for protein expression & purification. 6xHis-tag & Ni-NTA technology: The optimal partner for Strep-tag in double tag proteins  (pdf)
       IBA: Expression and purification of proteins using Strep-tag and/or 6xHistidine-tag. A comprehensive manual.   (pdf)
       IBA  Mammalian expression and purification system using Strep-tag and/or 6xHistidine-tag  (pdf) 
       JENA    6 % CL-Nickel ChroMatrix™ - Cobalt ChroMatrix™, Fast Flow and Nickel, Zinc, Cobalt, Copper and Metal Free Agaroses, Gravity Flow     (pdf)
       MERCK-NOVAGEN:  Metal Chelate Affinity Chromatography. Ni-MAC™, Co-MAC™ and u-MAC™ Metal Affinity Chromatography (MAC) Resins
            and Cartridges   (pdf)   (pdf-II)    (pdf-III)
       NOVAGEN:   pET purification manual   (pdf)   (pdf-2)   
       NOVAGEN:   Ni-NTA Hi-Bind Resins Protocols   (pdf)

       
NOVAGEN:   Protein Purification and Detection. His•Tag® Fusions (pg.18-29)   (pdf)
      
NOVAGEN:    His-Tag GST-Tag purification and Detection tools  (pdf)
      
NUNC:   ProPur™ affinity spin columns contain a unique flow regulator, FlowGo, which maximizes the contact time between the target protein and the resin.
           ProPur columns offer the speed of a spin column with the yields and purities of a gravity column or a batch protocol. They are idel for rapid
           small-scale and pilot purifications of His-tagged proteins, method development, expression trials, solubility determination and  separation of the proteolytically
           cleaved His tag from the purified protein.  (pdf-I)     
      
PIERCE:   HisGrab™ Nickel Coated 96-Well Plates. Ideal for analyzing polyhistidine-tagged fusion proteins by ELISA-based methods.
             Can be used with colorimetric, chemiluminescent or fluorescent detection methods. (pdf) 
      
PROMEGA:  MagneHisTM Protein Purification System   (pdf)  

        QIAGEN: Ni-NTA purification manual (pdf)  
        QIAGEN: NiNTA cell lysis under nature conditions (pdf)

        QIAGEN:   Ni-NTA Superflow Cartridges are pre-filled with 1 ml or 5 ml Ni-NTA Superflow and are ready to use for purification of 6xHis-tagged
             proteins using a syringe, peristaltic pump, or liquid chromatography system  (pdf)
        ROCHE     cOmplete His-Tag Purification Resin. Nickel-chelate chemistry that minimizes nickel ion leakage, and compatible with commonly used reducing
             agents (such as DTT), chelating metalloprotease inhibitors(such as EDTA), and a wide range of buffer substances and salt conditions.  (pdf-I)  (pdf-II)
       
SARTORIUS   Sartobind® IDA 75 - A Separation Technology Based on Metal Chelate Membrane Adsorbers - Operating Instructions   (pdf)
       
SIGMA:  His Selected Nickel Affinity Gel   (pdf)
        VIVASCIENCE:  Vivapure Metal Chelate Mini Spin Columns   (pdf-I)   (pdf-II)   (pdf-III)  (pdf-IV)   (pdf-V)   (pdf-VI)    (pdf-VII)    (pdf-VIII)  

Maltose Binding Protein
    GE-HEALTHCARE   MBPTrap™ HP is a ready to use HiTrap™ column for purifying recombinant proteins tagged with maltose
            binding protein (MBP). The column is packed with Dextrin Sepharose™ High Performance based on a 34 μm beadsized matrix. Purification of
            MBP-tagged protein is done under physiological conditions, which together with mild elution by maltose, preserves the activity of the target
            protein.  (pdf-I)   (pdf-II)      
    GE-HEALTHCARE  Purification of tagged proteins   (pdf)  
      NEW ENGLAND BIOLAB: pMAL protein Fusion and Purification System (pdf-I)    (pdf-II)
    
      PALL   Mixed-mode resins HEA HyperCel and PPA HyperCel can be used for MBP purification   (pdf)
       Small and large scale MBP-fusion protein purification
      D.R.Smyth et.al.: Crystal Structures of Fusion  Proteins with Large-Affinity tags. Protein Science (2003), 12: 1313-1322   (pdf)

Calmodulin Binding Peptide
     STRATAGENE: CBP Calmodulin-Binding Peptide Affinity Tag System   (pdf-I)   (pdf-II)   (pdf-III)

Intein Mediated Purification

     NEW ENGLAND BIOLABThe IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system is a novel protein        
             purification system which utilizes the inducible self-cleavage activity of protein splicing elements (termed inteins) to separate the target protein 
             from the affinity tag. Each intein tag contains a chitin binding domain (CBD) for the affinity purification of the fusion protein on a chitin resin. 
             Induction of on-column cleavage, using thiol reagents such as dithiothreitol (DTT), releases the target protein from the intein tag. 

             Able to produce target protein without vector–derived amino acids
 (pdf)
    
NEW ENGLAND BIOLAB: INTEIN-TM System   (pdf)
      NEW ENGLAND BIOLAB: INTEIN-TWIN System   (pdf)

T7.Tag
      NOVAGENT   7.Tag Affinity Purification kit   (pdf)  (pdf-II pg 45-46)

Cellulose Binding Domain
      NOVAGEN  CBIND Kits (pdf)

NusA Protein
      NOVAGEN   Expression of Soluble Heterologous Proteins via Fusion with NusA Protein - Article (pdf) 


Biotinylated Fusion Protein

        PROMEGA    PinPointTM Xa Protein Purification System  The System is designed for the production and purification of fusion proteins that
                are biotinylated in vivo. Biotinylated fusion proteins are produced in E. coli and are affinity-purified using the SoftLink™ Soft Release
                Avidin Resin. This proprietary resin allows elution of the fusion protein under nondenaturing conditions. The PinPoint™ Vectors
                feature the encoded endoproteinase Factor Xa  proteolytic site that provides a way to separate the purification tag from the native protein.
  (pdf) 
  

FLAG

        GenScript  DYKDDDDK or FLAG can be fused to the N-terminus or C-terminus of a target protein to facilitate detection and purification.
                Monoclonal Anti-DYKDDDDK G1 Affinity Resin  (pdf)
        SIGMA:  Detection and Purification - FLAG®  A Proven System for Detection and Purification of Proteins.  (pdf) 


Strep-Tag
  

        GE-HEALTHCARE    StrepTactin™ Sepharose™ High Performance is a chromatography medium for purifying Strep(II)-tagged proteins. Purification 
                is done under physiological conditions and mild elution preserves the activity of the target protein   (pdf)
        GE-HEALTHCARE  Purification of tagged proteins   (pdf)
        IBA:  Expression and purification of proteins using Strep-tag and/or 6xHistidine-tag. A comprehensive manual. The Strep-tag II is a short peptide
                 (8 amino acids, WSHPQFEK), which binds with high selectivity to Strep-Tactin, an engineered streptavidin. Elution of purified recombinant
                  protein is performed by desthiobiotin.
 (pdf)
        IBA  Tools for protein expression & purification. Strep-tag® technology and 6xHis-tag & Ni-NTA technology: Double tag protein expression
                 and purification
 (pdf)
        IBA  Mammalian expression and purification system using Strep-tag and/or 6xHistidine-tag  (pdf)
        IBA  One-STrEP Kit. One-STrEP-tag Purification for the Isolation and Identification of Protein Complexes in Mammalian Cells.
                 A comprehensive manual
 (pdf)
       NOVAGEN  Strep•Tactin® Purification Kits. Strep•Tactin protein is a streptavidin derivative developed for optimal Strep•Tag II binding. The
                 binding affinity of Strep•Tag II for Strep•Tactin is approximately 100 times higher than for streptavidin. The purified target protein is competitively
                 eluted with 2.5 mM desthiobiotin, an analog of biotin that reversibly binds Strep•Tactin.
  (pdf-I)  (pdf-II pg43-44)   (pdf-III)
       QIAGEN:   Two-Step Affinity Purification System Handbook - For expressing, purifying, and detecting proteins carrying a 6xHis
                 and Strep-tag II. After elution from the Ni-NTA matrix using imidazole, recombinant proteins (which also carry the Strep-tag II
                 epitope) are loaded directly onto a Strep-Tactin matrix and eluted using either biotin or desthiobiotin.
    (pdf-I)    (pdf-II)
        STRATAGENE:    VariFlex™ bacterial protein expression system. Include three different solubility enhancement tags (SETs) which are
                 designed to increase protein solubility, the streptavidin binding peptide (SBP) purification tag, and a tag that allows for rapid soluble protein
                 quantification (Q-tag)
   (pdf)

Fluorescent Protein
        BD BIOSCIENCES   BD Living Colors™ AcGFP1 Fluorescent Protein. A novel monomeric green fluorescent protein for fusion tag
                applications. Ideal for multicolor applications in flow cytometry and fluorescence microscopy.
 (pdf)   
        
BD BIOSCIENCES   BD Living Colors™ DsRed-Monomer Fluorescent Protein. A novel monomeric red fluorescent protein for fusion tag
                applications. Ideal for multicolor applications in flow cytometry and fluorescence microscopy.
(pdf)

Halo tag fusion

       PROMEGA:   The HaloTag™ Interchangeable Labeling Technology is a novel tool for imaging live or fixed mammalian cells that express the
                HaloTag™ Protein or protein fusions, analyzing post-translational modification of labeled fusion proteins, and isolating proteins and protein
                complexes. The technology is based on efficient formation of a covalent bond between a specially designed reporter protein encoded by the
                HaloTag™ pHT2 Vector and a specific ligand in living cells, in solution or on a solid support.  The HaloTag™ Ligand can carry a variety of
                functionalities, including fluorescent labels, affinity handles and attachments to a solid phase. The covalent bond forms rapidly under general
                physiological conditions, is highly specific and essentially irreversible, yielding a complex that is stable even under stringent conditions. The open
                architecture of the technology enables use of different ligands. Technical Manual.
(pdf)

       PROMEGA:   HaloLink™ Resin. The HaloTag technology comprises the HaloTag polypeptide, which can be fused to a protein of interest using
                the HaloTag Vectors, and a system of interchangeable synthetic ligands that covalently bind to the HaloTag polypeptide. HaloLink Resin, allows
                covalent and oriented surface immobilization of HaloTag fusion proteins. The HaloLink Resin can be used in a variety of applications including
                enzyme immobilization, detection of protein:protein interactions and purification of fusion proteins using protease cleavage.
 (pdf)

   Solubility enhancement tags (SETs)

        STRATAGENE:    VariFlex™ bacterial protein expression system. Include three different solubility enhancement tags (SETs) which are
                  designed to increase protein solubility, the streptavidin binding peptide (SBP) purification tag, and a tag that allows for rapid soluble protein
                  quantification (Q-tag)
   (pdf)

  SUMO

        INVITROGEN:   The Champion™ pET SUMO Protein Expression System utilizes a small ubiquitinlike modifier (SUMO) to allow expression,
                 purification, and generation of native proteins in E. coli. SUMO fusions may increase the expression of recombinant proteins and enhance the
                 solubilityof partially insoluble proteins. In addition, the tertiary structure of the SUMO protein is specifically recognized and cleaved by a
                 ubiquitin-like protein-processing enzyme, SUMO Protease resulting in the production of native protein.
 (pdf)
        LIFESENSORS  SUMO Gene Fusion Technology  (pdf)
        LIFESENSORS  SUMO Gene Fusion Technology - New Methods for Enhancing Protein Expression and Purification in Bacteria   (pdf-I)   (pdf-II) 
                (pdf-III)
        LIFESENSORS  SUMO Gene Fusion Technology - New Methods for Enhancing Protein Expression and Purification (intracellular and secretory
                expression)
in Insect cells (pdf)    
       
LIFESENSORS  SUMO Gene Fusion Technology - New Methods for Enhancing Protein Expression and Purification (intracellular and secretory
                expression) in Mammalian cells
 (pdf)  
       
LIFESENSORS  SUMO Gene Fusion Technology - New Methods for Enhancing Protein Expression and Purification in Yeast cells
                Saccharomyces (pdf) and Pichia (pdf)    
       LIFESENSORS  SUMO Gene Fusion Technology - New Methods for Rapid Recombinant Peptide Expression and Purification   (pdf)


Cold Expression

          TAKARA  pCold TF DNA Vector is a fusion cold shock expression vector that expresses Trigger Factor (TF) chaperone as a soluble tag
                    (48 kDa) which facilitates co-translational folding of newly expressed polypeptides. The vector has a His-Tag sequence, and a multicloning site
                    (MCS). A lac operator is inserted downstream of  the cspA promoter to ensure strict regulation of expression. Additionally, recognition sites
                    for HRV 3C Protease, Thrombin, and Factor Xa are located between TF-Tag and the Multiple Cloning Site (MCS) and function to facilitate
                    tag removal from the expressed fusion protein. Most E. coli strains can serve as expression hosts. The pCold TF DNA Vector provides cold
                    shock technology for high yield protein expression combined with Trigger Factor (chaperone) expression to facilitate correct protein folding,
                    thus enabling efficient soluble protein production for otherwise intractable target proteins.   (pdf-I)   Chaperone Plasmid Set / Chaperone
                    Competent Cells
(pdf-II)    

LYTAG and LYTAG Two Phase System

             BIOMEDAL    LYTAG system allows the single-step purification of proteins fusion to LYTAG and C-LYTAG using LYTRAP resin, a 
                    simple and efficient chromatographic support. Binding conditions are gentle and do not involve covalent modifications, in such a way that the fusion 
                    protein is highly stable once bound to the resin. LYTAG and C-LYTAG fusions protein are selectively eluted using choline-containing buffers. 
                    No unwanted amino acids after tag elimination by enterokinase digestion  (pdf)

             BIOMEDAL    LYTAG Two-Phase is a protein purification system based on the use of two aqueous components. 
                    The method relies on the affinity of the protein tag LYTAG for one of the two-phase components, allowing recombinant protein separation 
                    and purification from cellular extracts or culture media. In the procedure, the LYTAG-fused protein is retained in one of the aqueous phases while most 
                    of the undesired proteins can be removed by simply discarding the opposite phase. After replenishing the system with fresh phase, the protein of interest 
                    can be easily recovered in it, with high purity, by reversing its localization with the addition of choline, the specific LYTAG ligand. Purification can be easy 
                    and safely performed at low temperatures, requiring only a refrigerated centrifuge and an ice water bath. Alternative to conventional solid resins.  (pdf-I) 
                    (pdf-II)

Profinity eXact™

            BIO-RAD   Profinity eXact fusion-tag system is an affinity tag-based protein purification system that  utilizes a modified form of the subtilisin
                    protease, which is immobilized onto a chromatographic support and used to generate pure, tag-free target protein in a single step.
                    The tag in this system is the prodomain (prosignal sequence) of the subtilisin protease, a 75-amino acid sequence that is fused to the
                    N-terminus of a target protein of interest. The mature and prodomain subtilisin protease sequences have been co-engineered to produce a
                    highly specific, high-affinity interaction between the binding partners. Application of elution buffer triggers subtilisin’s processing activity,
                    which quickly and precisely cleaves the tag from the fusion protein and releases the purified target protein. At the end of the purification
                    process, the tag remains tightly bound to the resin and contains only its native amino acid sequence. The structure and activity of the native
                    protein is maintained, and the need for additional materials, such as cleavage enzymes and purification resin for postcleavage enzyme removal,
                    is eliminated.  (pdf-I)  (pdf-II)   (pdf-III)  Profinity eXact purification resin  (pdf-IV)   (pdf-V)

Bispecific Affinity

        Johan Nilvebrant, Tove Alm, Sophia Hober (2012)   Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
            Journal of Visualized Experiments 59 e3370: 1-5          
            Bispecific purification tag with two different binding sites on a 46 amino acid small protein domain: an albumin-binding domain (derived from
            Streptococcal protein G and with a strong affinity to human serum albumin (HSA), that can be recognized with HSA Sepharose column; and
            a second domain against a dimeric Z-domain derived from Staphylococcal protein A, that can bound HiTrap MabSelect SuRe from GE   (pdf)

            The video component of this article can be found at http://www.jove.com/video/3370/

CaptureSelect C‐tag

            CaptureSelect   CaptureSelect C‐tag. A small 4 amino acid peptide tag: E‐P‐E‐A (glutamic acid ‐ proline ‐ glutamic acid ‐ alanine)
                    that binds to a 13 kDa Camelid antibody fragment affinity matrix. The CaptureSelect C‐tag affinity matrix purifies C‐terminal tagged
                    proteins with high affinity and selectivity, even in the presence of Urea and Guanidine HCl, from complex mixtures like cytoplasm or
                    periplasmatic fractions in a one step process. Mild elution conditions at neutral pH can be applied using 2M magnesium chloride or 50%
                    propylene glycol, which ensures high activity recoveries of pH sensitive target proteins; or more specifically with 2mM S‐E‐P‐E‐A
                    peptides. The affinity resin recognizes the E‐P‐E‐A tag sequence when fused either directly to the C‐terminus of a protein or through
                    linkers between the C‐terminus and the E‐P‐E‐A tag   (pdf)

Cleavage of Recombinant Proteins

Cleavage Sites Table for Fusion Proteins

    Proteases sensitivity to Detergents

          James M. Vergis, Michael C. Wiener (2011)    The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal
            Protein Expression and Purification 78: 139–142  (pdf)
          David S. Waugh  (2011)    An overview of enzymatic reagents for the removal of affinity tags
           
Protein Expression and Purification  (pdf)
          Mohanty A. et.al. (2003) Inhibition of tobacco etch virus protease activity by detergents. Protein Expression and Purification 27: 109–114  (pdf)

Factor Xa
       Factor Xa Cleavage of a MBP fusion protein
          NOVAGEN: Factor-Xa kit  (pdf)
          NOVAGEN:  Restriction Grade Factor Xa. Factor Xa Cleavage Capture Kit .  (pdf pg.54)
          QIAGEN:  Factor Xa  and Factor Xa Removal resin   (pdf)

Thrombin
       Thrombin Cleavage of a GST fusion protein
         NOVAGEN:  Thrombin kit    (pdf) 
         NOVAGEN:  Thrombin, Restriction Grade. Biotinylated Thrombin and Thrombin Cleavage Capture Kit  (pdf pg.53)

Enterokinase
       NEW ENGLAND BIOLABS:   Enterokinase  (pdf)
         NOVAGEN:  Enterokinase cleavage capture kit   (pdf)
       NOVAGEN:  Recombinant Enterokinase. Tag•off™ High Activity rEK is a highly purified preparation of the catalytic subunit of human enterokinase 
             that recognizes the identical cleavage site as the native enzyme, AspAspAspAspLys↓. Enterokinase Cleavage Capture Kit. Tag•off™ rEK Cleavage 
             Capture Kit  (pdf pg.55-56)

      
ROCHE:    Enterokinase   (pdf)

PreScission
       GE-HEALTHCARE   Application Note   (pdf)
         GE-HEALTHCARE   Life Science News   (pdf)

TAGZyme
     
UNIZYME  The TAGZyme System is an efficient and specific method for complete removal of N-terminal his-tags by use of exopeptidases. 
            The method is based on the use of dipeptidyl aminopeptidase I (DAPase) alone or in combination with glutamine cyclotransferase (Qcyclase)
            and pyroglutamyl aminopeptidase (pGAPase). These enzymes all have the ability to bind to IMAC matrices through an engineered his-tag in 
            recombinant forms
     QIAGEN:  His-tag Removal by exoproteolytic Digestion   (pdf)
     José Arnau, et.al. (2006)  Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins
            Protein Expression and Purification 48 1–13  (pdf)

TEV-Protease
         INVITROGEN- LIFE TECHNOLOGIES  TEV-Protease(pdf) 
        Mohanty A. et.al. (2003) Inhibition of tobacco etch virus protease activity by detergents. Protein Expression and Purification 27: 109–114  (pdf)
        PROMEGA:  ProTEV Protease is an improved 50kDa version of the NIa protease from tobacco etch virus (TEV) that has been engineered to be
            more stable than native TEV protease for prolonged enzymatic activity  (pdf)
        CARDIFF UNIVERSITY - MOLECULAR CELL BIOLOGY - EHRMAN LAB   TEV NIa protease

HRV 3C Protease

        NOVAGEN    HRV 3C Protease   (pdf)    (pdf pg.57)   

SUMO   

        INVITROGEN   SUMO Protease  (pdf)   
        LIFESENSORS  SUMO Protease 1   (pdf)    (pdf-II)   (pdf-III)

Affinity Chromatography 

   Activated Resins
   
GE-HEALTHCARE   Affinity Manual  (pdf)
      GE-HEALTHCARE   Affinity Columns and Media  (pdf)  
            HiTrap NHS-activated HP, NHS-activated Sepharose 4 Fast Flow (10-atom spacer arms) is designed for the covalent coupling
            through the primary amine of a ligand and is the first choice for the preparation of immunospecific media.
            CNBr-activated Sepharose offers a well-established option for the attachment of larger ligands and as an alternative to NHS-activated
            Sepharose. Proteins, peptides, amino acids or nucleic acids can be coupled to CNBr-activated Sepharose, under mild conditions, via
            primary amino groups or similar nucleophilic groups.
            EAH Sepharose  4B for coupling small ligands containing free acarboxyl groups via a 10-atom spacer arm. Carbodiimide as the coupling
            reagent. Phenolic groups may be attached through others couple reagents. Thiol derivatives can couple carboxyl groups in the presence of
            carbodiimide and the thiol ester bond may be cleaved specifically using hydroxylamine, thus providing a simple and gentle method for
            eluting the intact ligand-protein complex.
            ECH Sepharose 4B for coupling small ligands containing free amino groups via a 9-atom spacer arm. Carbodiimide as the coupling reagent.
            Epoxy-activated Sepharose 6B for coupling through hydroxy, amino or thiol groups via a 12-carbon spacer arm. It is particularly useful for
            coupling small ligands such as choline, ethanolamine and sugars. The pre-activated matrix is formed by reacting Sepharose 6B with the
            bis oxirane, 1,4 bis-(2,3-epoxypropoxy-)butane. Free oxirane groups couple via stable ether bonds with hydroxyl-containing molecules
            such as sugars, via alkylamine linkages with ligands containing amino groups, and via thioether linkages with ligands containing thiol groups.
            Thiopropyl Sepharose 6B for coupling through a thiol group many types of small ligands
                    • Heavy metal ions and derivatives can be used as ligands to react with thiol groups forming mercaptides.
                    • Alkyl or aryl halide ligands give thioether derivatives.
                    • Ligands containing C=O, N=N and, under certain conditions, C=C bonds undergo addition reactions.
            The hydroxypropyl group acts as a small spacer arm. Ligands containing amino groups can be attached to Thiopropyl Sepharose 6B
            or Activated Thiol-Sepharose 4B by multi-point attachment or coupling through a small number of groups using the heterobifunctional
            thiolating reagent, SPDP. The coupled molecules may be recovered by eluting with a reducing agent.

      GE-HEALTHCARE   CNBr Activated Sepharose Data File  - React with primary amines present on proteins, antibodies
            and other molecules.
  (pdf)
      GE-HEALTHCARE   NHS Activated Sepharose Data File -NHS (N-hydroxysuccinimide) coupling forms a chemically
            stable amide bond with ligands containing primary amino groups. NHS-activated Sepharose® 4 Fast Flow provides a spacer arm
            and is therefore particularly suitable for immobilising small protein and peptide ligands.
  (pdf)   
      GE-HEALTHCARE   Recommended carbodiimide coupling procedure for CH Sepharose® 4B and AH Sepharose® 4B  (pdf)
      BIO RAD   Affi-Gel 10 and Affi-Gel 15 activated immunoaffinity supports (N-hydroxysuccinimide esters of a derivatized crosslinked agarose),
            with a 10 and 15-atom spacer arm respectively, that offer rapid, high efficiency coupling for all ligands with a primary amino group aqueous
            or non-aqueous solution
.
     (pdf)
      ABT  Amino Ethyl and Glyoxal Agarose Beads (pdf-I)    (pdf-II)
            Glyoxal Agarose Beads  includes a broad selection of resins for the immobilization of biomolecules through amino groups (enzymes,
            antibodies, etc). There are resins, with either 4% or 6% agarose concentration, and with various degrees of activation (very high/ high/low).
            This permits the immobilization of biomolecules in different size ranges.
(pdf-I)
            Aminoethyl Agarose Beads covalently bind the agarose to the acid groups of the amino acid of the target biomolecules: acidic amino acids 
            like aspartic acid or glutamic acid.There are resins with agarose concentration of 4% or 6% and with a range of degrees of activation.

            (pdf-I)  

      JENA BIOSCIENCE   Immobilized Nucleotides for Affinity Chromatography   (pdf)   Immobilized nucleotides provide a convenient
            and rapid one-step purification procedure for a large number of proteins such as kinases, GTPases, chaperones, motor proteins, and others.
            Jena Bioscience provides NTPs and dNTPs that are linked to the matrix at various positions of sugar, base or phosphate moiety of the
            nucleotide; different types and lengths of linkers and different types of chromatography material ranging from bulk material to pre-made
            columns that fit any machine

     
MERCK  Fractogel EMD  Amino   (pdf)   For immobilization of ligands with carboxy-functionional groups.
      MERCK  Fractogel EMD  Epoxy (M)  (pdf)   Suitable for coupling of low molecular weight ligands and pH stable proteins. The epoxyde reacts
            with primary amino groups, hydroxyl, and sulfhydryl groups. The resulting affinity matrix is very stable, due to the ether bonding of the ligand.

      NOVAGEN  Preactivated Resins  (pdf) 

           PreACT™ Agarose ALD is an activated chromatography matrix for simple and efficient immobilization of small ligands to large
            proteins under mild physiological conditions. Is supplied in an activated form containing bound reactive aldehyde groups. During
            ligand coupling, aldehydes react with primary amines on the ligand to form a Schiff-base. In a further reaction, the Schiff-base linkage
            is selectively converted into a stable, secondary amine linkage.
            PreACT Fractogel AZL is an activated resin with a high density of tentacle-bonded azlactone reactive groups suitable for the
            coupling of proteins under physiological conditions.  The azlactone ring is opened by nucleophilic attack of appropriate groups
            present on the protein surface. Upon reaction with amines, a stable amide bond forms between the former carbonyl function of the azlactone.
            PreACT Fractogel EPX is an activated resin for the immobilization of low molecular weight amine bearing ligands or alkaline-stable proteins.
            Bonded epoxide groups react with primary amino, hydroxyl and sulfhydryl groups to form stable ether linkages.

      PIERCE:   Affinity Purification Handbook  (pdf)
      PIERCE   Coupling different Functional Groups: Tosyl, Tresyl and Epoxy Activated Agarose. Alkylamine Beads
            Tosyl Activated Agarose Hydroxyls on the surface of cross-linked beaded agarose are reacted with p-toluenesulfonyl chloride (tosyl chloride)
            to yield a sulfonated support. These sulfonates can couple to nucleophiles, such as primary amines or thiols, to yield a stable affinity support.
            The tosylated support also couples to imidazole, or tyrosine hydroxyl groups. Tosyl Activated Agarose couples more rapidly and more efficiently
            to sulfhydryl groups than to primary amines. Sulfhydryl coupling supports are convenient when accessible free sulfhydryls exist in the protein, or
            when one can be easily generated through reduction of a disulfide bond.
            Tresyl Activated Agarose Hydroxyls on the surface of agarose are reacted with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) to yield
            a sulfonated support.    This sulfonated support is approximately 100-fold more reactive than a Tosyl agarose support. Tresyl Activated Agarose
            can couple to nucleophiles, such as primary amines or thiols, to yield a stable affinity matrix.. The tresylated support also couples to imidazole
            and tyrosine hydroxyl groups. Tresyl Activated Agarose couples more rapidly and more efficiently to sulfhydryl groups than to primary amines.
            ImmunoPure® Epoxy-Activated Agarose Epoxide chemistry is a useful way to immobilize ligands  like proteins, carbohydrates, peptides and
            amino acids, containing nucleophiles, such as amino,thiol and hydroxyl (including phenolic) functional groups. Epoxide-activated supports are
            produced by the immobilization of bifunctional oxiranes such as 1,4-butanediol diglycidyl ether onto agarose supports, introducing a hydrophilic
            spacer arm. These activated supports have limited stability in aqueous media, so it is necessary to use them quickly after they are generated or
            rehydrated.
  (pdf)
      PIERCE   CarboLink® Coupling Gel allows immobilization of glycoproteins through their oxidized carbohydrate moieties. Because
            carbohydrates are located on the Fc portion of antibody molecules, CarboLink® Gel has the advantage of orienting the antibody
            binding sites to remain unobstructed, resulting in greater purification capability.
  (pdf)
      PIERCE   Coupling Proteins trough Sulfhydril groups  Pierce SulfoLink® Coupling Gel is designed to efficiently react with thiol-containing
            molecules and immobilize them through a thioether linkage. The support contains an iodoacetyl group at the end of a long spacer arm,
            which reacts with sulfhydryls through displacement of the iodine.
  (pdf)
      PIERCE:    The AminoLink® Coupling Gel support is 4% cross-linked beaded agarose that has been activated to form aldehyde
            functional groups. The aldehydes react spontaneously with primary amines found in lysine residues and at the amino terminus of a
            peptide chain. Reductive amination of the resulting Schiff base then forms a stable, secondary amine linkage with minimal leakage
            of the ligand.
  (pdf)
      PIERCE:   Batch and Spin Cup Methods for Affinity Purification of Proteins  (pdf)   
      PIERCE:   MicroLink™ Protein Coupling Kit allows immobilization of small amounts (25-100 µg) of purified antibody and other
            proteins directly onto beaded agarose gel to create a permanent affinity support. The AminoLink® Plus Coupling Gel included
            in this kit contains aldehyde functional groups that react with primary amines present on antibodies and other molecules.
            Reductive amination of the resulting Schiff bases forms a stable secondary amine linkage with minimal leakage. 
(pdf)
      PIERCE:   MicroLink™ Peptide Coupling Kit. For sulfhydryl-containing peptide or protein. This kit uses UltraLink® Iodoacetyl Gel
            that reacts specifically with free sulfhydryls to form a stable thioether linkage. The support contains a 15-atom spacer that reduces
            steric hindrance, making binding interactions with the coupled molecule efficient. 
(pdf)
      PIERCE:   Ultra Link Immobilized Carboxy for Inmobilization of Peptides using EDC. Useful for coupling primary amine containing ligands
            like small peptides
 (pdf)
      PIERCE:   UltraLink® Iodoacetyl Gel binds specifically to sulfhydryls when used under specific conditions with the iodoacetyl alkylating agent. 
            The 15-atom spacer arm is especially ideal for conjugating small peptides to the support. The support will immobilize numerous types of
            molecules with a free sulfhydryl including antibodies, other proteins and peptides.
  (pdf)
      PIERCE:   Ultra Link Biosupport Medium with Azlactone Groups. Couples nucleophiles on ligands via a ring opening reaction to
            attach the ligand to the support through stable covalent linkages. Amino-functional ligands will form stable amide bonds at the end
            of a five-atom spacer.
   (pdf)
      SARTORIUS  Sartobind® Epoxy 75 - A Microporous Coupling Membrane for Affinity Chromatography - Operating Instructions -
            Any molecule containing amino-, hydroxyl- or thiol-groups may be immobilized by covalent coupling to the epoxy-activated membrane.
            The membrane is fitted into a filter holder with Luer Lock connectors for easy handling to quickly couple biomolecules like proteins
            or peptides covalently.
  (pdf)
      TOSOH   Affinity Chromatography Activated Resins   (pdf) 
            TOYOPEARL AF-Tresyl-650M:  For coupling of proteins through amine and thiol groups at slightly alkaline pH (7.0-8.0)
            TOYOPEARL AF-Epoxy-650M: Useful for attaching low MW ligands at high densities and for immobilization of carbohydrates or glycoproteins
            TOYOPEARL AF-Formyl-650M: (aldehide bearing)
For coupling of proteins through amine groups at slightly alkaline pH (7.0-9.0)  
            TOYOPEARL AF-Amino-650M
:  For coupling ligands through their carboxylate groups (peptide bond formation) or aldehyde groups
            (reductive amination) present in carbohydrates and glycoprotein ligands, or introduced into the ligand by mild periodate oxidation. Reaction at
            slightly acidic pH (4.5-6.0)  
            TOYOPEARL AF-Amino-650M
: Carbodiimide mediated coupling reaction to amino groups of proteins or low MW ligands. Reaction at
            slightly acidic pH (4.5-6.0)
 
      VIVASCIENCE:  Vivapure Epoxy Coupling Kit - Mini Spin Columns - Vivapure Epoxy Mini spin columns provide a membrane to
            which any desired protein, containing amino-, sulfhydryl-, or hydroxyl-groups on its surface is coupled covalently.
  (pdf)
     
 
       Antibody Purification and antibody fragmentation
       

        GE-HEALTHCARE   Antibody  Purification Manual   (old pdf)   (new pdf) 
        GE-HEALTHCARE   Application Note: Rapid optimisation and development of an automated two-step purification procedure for
                           monoclonal IgG antibodies
  (pdf)   
        GE-HEALTHCARE   Capto adhere: a strong anion exchanger with multimodal functionality ionic interaction, hydrogen bonding and
             hydrophobic interaction. Capto adhere is designed for post Protein A purification of monoclonal antibodies. Removal of leached Protein A,
            aggregates, host cell proteins, nucleic acids and viruses from monoclonal antibodies is performed in flowthrough mode at where the antibodies
            pass directly through the column while the contaminants are adsorbed.  (pdf-I)  (pdf-II)  (pdf-III)
        GE-HEALTHCARE   Kappa Select is an affinity chromatography medium designed for the purification of Fab (kappa) with high binding capacity, purity
                        and yield. The ligand binds to the constant region of the kappa light chain. Use for the production of Fabs for clinical applications. (pdf)
        GE-HEALTHCARE   HiTrap MabSelect SuRe, HiTrap MabSelect and HiTrap MabSelect Xtra for purification of monoclonal antibodies (MAbs(pdf)
        BAC (BioAffinity Company)  CaptureSelect Human IgA affinity matrix contains an affinity ligand that binds to a unique domain that is present on all    
            subclasses of human IgA, with no cross reactivity with IgM or IgG. (pdf-I)   (pdf-II)
            CaptureSelect IgM affinity matrix contains an affinity ligand that is directed towards a unique domain that is present on both human and mouse IgM  
            antibodies, and offers no cross-reactivity with human or mouse IgA or IgM. (pdf)
            CaptureSelect Fab kappa affinity matrix: for all human antibodies containing kappa light chains  (pdf)
            CaptureSelect Fab lambda affinity matrix: for all human antibodies containing lambda light chains  (pdf)
            CaptureSelect Human Fc affinity matrix: for all subclasses of human IgG  (pdf)
            CaptureSelect Multi Species affinity matrix: for IgG from multiple species  (pdf)
            CaptureSelect Multi Species affinity matrix: for human IgG4  (pdf)  
        BIORAD    Affi-Prep® Protein A Matrix Instruction Manual   (pdf)
        BIORAD    Affi-Gel® Protein A MAPS® II Kit - Instruction Manual  (pdf)
        
BIORAD    Econo-Pac® Protein A Kit Protein A Columns - Instruction Manual  (pdf)
        BIO RAD   Immunoaffinity Kit for IgG coupling trough Fc region    (pdf)
        BIO RAD   Protein A removal  from IgG on CHT Ceramic Hydroxyapatite Support   (pdf)
        CALBIOCHEM   Human IgG subclasses   (pdf)
   
        CLONTECH: Thiophilic - Purification of Immunoglobulins (pdf) 
        GENOVIS:    FabRICATOR® is the fastest and most accurate enzymatic tool to generate F(ab’)2 and Fab fragments from antibodies. 
            The FabRICATOR® utilizes the unique specificity of a novel proteolytic enzyme that cleaves all human, rabbit, sheep and monkey IgG
            subclasses at the same place in the hinge region creating pure F(ab’)2 fragments without any further degradation. Cleave IgG within
            just a few minutes, at mild, physiological pH conditions.
            No sample degradation, high yields.  (pdf)FragIT™ columns consist of the proteolytic antibody-specific enzyme FabRICATOR immobilized
            on agarose. FragIT™ is the fastest and easiest  way to fragment antibodies into F(ab')2 and Fc; cleave up to 100mg IgG in just half an hour.
            Reaction is carried out at pH 6.8.
 (pdf) 
            FragIT™ microspin columns allow for easy and quick fragmentation of IgG into F(ab’)2 and Fc fragment; fragmentation of up to 0.5 mg IgG in less
            than  30 minutes.
            FragIT™ microspin columns come prefilled with FabRICATOR® enzyme immobilized on agarose
 (pdf)
            IgGZERO™ is a very efficient endoglucosidase with high specificity for IgG. The enzyme cleaves the chitobiose core of the glycan on IgG; it has the
            ability to hydrolyze human, Rhesus monkey, mouse, rat, rabbit, horse, goat, dog and porcine native and denaturated IgG.Removal of IgGZERO™ is
            extreamly  easy by the included histidine tag in the amino terminal of the recombinant enzyme  (pdf-I)  Micrspin  (pdf-II)  
        MERCK   Capture of Mouse Monoclonal Antibodies by Strong Cation Exchange Chromatography    (pdf)  
                  Eshmuno™ S, a cation exchanger specifically designed for highly productive downstream purification of monoclonal antibodies. Hydrophilic 
                   polyvinyl ether base matrix that allows the use of much higher flow rates.  (pdf)
        MILLIPORE   Affinity Chromatography Media: ProSep®-vA Ultra Media, ProSep-vA High Capacity Media, ProSep-A High Capacity Media &
                 ProSep-rA High Capacity Media. OPERATING INSTRUCTIONS ProSep-A affinity media have been developed specifically for industrial
                 scale purification of monoclonal antibodies where highly efficient purification can be achieved using clarified bioreactor feedstock at physiological
                 pH and salt concentration.
(pdf)
        MILLIPORE  PROSEP-Thiosorb and PROSEP-Thiosorb M Chromatography Media - for the recovery and purification of Immunoglobulins  (pdf)
        
Pall         BioSepra® Protein A Ceramic HyperD® F sorbent. High capacity affinity sorbent designed for process-scale purification of
                  immunoglobulins G. (pdf)      (pdf-II)
        Pall         Purification of  mouse IgM from cell culture supernatant by Cation Exchange Chromatography on CM Ceramic HyperD F sorbent  (pdf)
       
Pall         Purification of mIgG1 on MEP HyperCel Mixed-Mode media. Optimization  (pdf)
        PIERCE:   Antibody Technical Handbook. The handbook provides an overview of antibody structure and types, as well as technical information
                   on the procedures, reagents and tools used to produce, purify, fragment and label antibodies.  (pdf-1)  (pdf-2)  (pdf-3)   
        PIERCE:   Affinity Purification Handbook  (pdf)
        PIERCE:  Binding Characteristics of Immunoglobulin Binding Proteins and Thiophilic Gel  (PowerPoint)
        PIERCE:  Fab Preparation Kit enables efficient Fab generation from IgG. This kit uses papain, a nonspecific thiol-endopeptidase,  immobilized
            on agarose resin. Immobilized enzyme is advantageous because digestion can be immediately stopped by simply removing the IgG solution
            from the resin, resulting in a digest that is enzyme-free. Digestion by papain produces 50 kDa Fab and Fc fragments  (pdf)
        PIERCE:  Immobilized E. coli Lysate. Partially purified bacterial proteins from a suspension of E. coli cells (strain BMH 71-18) immobilized onto
                        agarose, provides a simple and efficient method of removing E. coli-reactive proteins from antibody preparations
 (pdf_I)   (pdf-II)
        PIERCE:  
Antibody Immobilization: Choosing the Best Support   (PowerPoint)
        PIERCE:  Immobilized Jacalin (human IgA and IgD binding lectin).
                    Jacalin immobilized on supports such as agarose has been useful for the purification of human serum or secratory IgA1
   (pdf)    (pdf-II)
        PIERCE:  T-Gel™ Purification Kit  (pdf)   
        PIERCE:  Pierce® Protein L Agarose. Protein L binds to immunoglobulin kappa light chains without interfering with the antigen-binding site
                and binds a wider range of Ig classes and subclasses than other antibody-binding proteins such as Protein A or Protein G. Protein L
                binds to all classes of Ig (i.e., IgG, IgM, IgA, IgE and IgD). Protein L also binds single chain variable fragments (Scfv) and Fab
                fragments. Protein L only binds to immunoglobulins containing light chains of type kappa I, III, and IV in human and kappa I in mouse.
                Protein L also may be specific for certain kappa subgroups in other species. Protein L binds scfv without interfering with antigen binding.
                Protein L binds weakly to rabbit immunoglobulins and does not bind immunoglobulins from bovine, goat or sheep; nor does it bind to
                lambda light chains.  (pdf)
       
SARTORIUS  Sartobind Protein A 75 Membrane Adsorbers - Operating Instructions 
                  A Separation Technology Based on Microporous Membranes
 (pdf)
       VIVASCIENCE: Vivapure Protein A - Mini Spin Columns (pdf) 

       General

       BIO RAD   Polymixin matrix - For removal of Endotoxin molecules (pdf) 
       
GE-HEALTHCARE    VIII Select. Highly selective to recombinant b-domain depleted Factor VIII a key recombinant blood factor used for
               treatment of Hemophilia A. Efficient purification of recombinant b-domain.   (pdf)
       
GE-HEALTHCARE   Kappa Select is an affinity chromatography medium designed for the purification of Fab (kappa) with high binding capacity,
              purity and yield. The ligand binds to the constant region of the kappa light chain. Use for the production of Fabs for clinical applications. (pdf)
       MILLIPORE  Matrex Cellufine Sulfate - For concentration, purification and depyrogenation of Virus, Viral/Microbial Antigen,
                Heparin binding proteins
 (pdf)   
        Pall  
BioSepra Blue Trisacryl® M is an affinity chromatographic sorbent used for the purification of a wide variety of enzymes and proteins such
               as kinases, albumin, interferons, and some coagulation factors.  (pdf)
        Pall  BioSepra® Heparin HyperD® M composite chromatography sorbent for the purification of biological molecules that bind to heparin,
                such as coagulation factors, growth factors, lipoproteins. (pdf) 

    
     
Immunoprecipitation

         ABCAM:  Immunoprecipitation protocol. 1. Lysis buffers and other reagents 2. Preparation of lysates 3. Pre-clearing the lysates 4.
                Immunoprecipitation  5. Choosing the correct beads   (pdf-1)   Troubleshhoting   (pdf-II)   Procedure to cross link the antibody to
                the beads to enable elution of protein with little antibody contamination  (pdf-III)  
        INVITROGEN:  Dynabeads® for protein complex isolation. Add your specific antibody or interacting protein with tags to Dynabeads®,
                then immunoprecipitate your protein of interest. Once the beads are exposed to a magnet, they are efficiently drawn to the tube wall,
                taking only your protein complex with them. As the process is gentle, yet very quick, complexes remain intact and functional. The
                complexes can be resuspended in a small volume ready for downstream analysis with mass spectrometry, gels, etc.  (pdf)
         INVITROGEN:  Immunoprecipitation with Dynabeads® Protein A or Protein G. The captured immune complexes are easily removed from
               the supernatant by  magnetic separation.  (pdf)
         PIERCE:  Seize™ X Immunoprecipitation Kits. Recover protein without antibody protein band interference. The Kit combine cross-linking
                and affinity chromatography to offer a new and improved immunoprecipitation method. First, the primary antibody is bound and immobilized to
                a Protein A or Protein G support using cross-linking agent (DSS). This properly orients the antibody to "seize" protein from crude cell lysate
                applied to the immobilized antibody support. Unbound proteins are then centrifuged away and the protein is recovered by using an elution buffer.
                (pdf -I)  (pdf-II)  (pdf-III)  (pdf-IV)
         PIERCE:   The Thermo Scientific Pierce Direct IP Kit enables highly effective and efficient antigen immunoprecipitations by directly immobilizing
                purified antibodies onto an agarose support. Immobilizing the antibody provides faster and easier immunoprecipitations, enables reuse of the
                immobilized antibody and results in purified antigen free from antibody contamination. Immunoprecipitation is achieved using less than 10 μg of
                antibody and a short coupling protocol to AminoLink Resin. The antigen sample is incubated with the immobilized antibody to form the immune
                complex. The complex is washed to remove non-bound material, and a low pH elution buffer is used to dissociate the bound antigen from the
                antibody.  (pdf)  


       Lectins and Glycoprotein Purification
    
       
CLONTECH:   Glycoprotein Enrichment Resin is a phenylboronic acid-based resin which provides quick, efficient, and specific enrichment
              of glycoproteins  from complex samples such as human serum. The resin consists of an m-aminophenylboronic acid ligand coupled to
              agarose beads. The ligand binds to cis-diol groups on sugar residues such as mannose, galactose, or glucose that are present within the
              saccharide moiety of glycoprotein molecules.
              This complex can be dissociated by lowering the pH, or by using an elution buffer containing either Tris or sorbitol.  (pdf)
       
GE-HEALTHCARE  Con A Sepharose 4B  (Con A binds molcules containing Alpha-D-mannopyranosyl, Alpha-D-glucopyranosyl
             and sterically related residues)
 (pdf-I)    (pdf-II) 
       
GE-HEALTHCARE  HiTrap Lectin Test Kit consists of four glycoprotein binding columns, HiTrap Con A, HiTrap Lentil Lectin,
               HiTrap Wheat Germ Lectin and HiTrap Peanut Lectin
.  (pdf)

       
GE-HEALTHCARE   HiTrap Wheat Germ Lectin (high affinity to N-acetylglucosamine and reacts strongly with the chitobiose core of N-linked
             oligosaccharides and N-acetylneuraminic acid)
 (pdf)   
       
GE-HEALTHCARE  Lentil Lectin Sepharose 4B (binds to polysaccharides and glycoconjugates containing glucose or mannose
               type sugars)
  (pdf)
        CALBIOCHEM   Lectins  (pdf) 
    
EY Laboratories Lectin Catalog. Lectin carbohydrate binding chart. Immobilized lectins and carbohydrates. Lectins kits.  (pdf)
    
PIERCE:   Immobilized Jacalin (human IgA and IgD binding lectin). Jacalin immobilized on supports such as agarose has been useful for the
               purification of human serum or secratory IgA1
   (pdf)    (pdf-II)   
        PIERCE:   Glycoprotein Isolation Kit, WGA isolates glycoproteins from complex protein mixtures using the lectin wheat germ agglutinin (WGA)
                immobilized on agarose. The WGA lectin preferentially binds N-acetyl glucosamine (GlcNAC) and terminal GlcNAC structures that
                are commonly present in many serum and membrane glycoproteins. WGA also has affinity for sialic acid.
 (pdf)  
        PIERCE:   Glycoprotein Isolation Kit, ConA isolates glycoproteins from complex protein mixtures using the lectin concanavalin A (ConA)
               immobilized on agarose. The ConA lectin preferentially recognizes α-linked mannose and, to a lesser extent, terminal glucose residues
(pdf)  
        PROMETIC BIOSCIENCES   Aminophenyl Boronate Affinity Chromatography for capture of glycoproteins. The aminophenyl
                boronate-glycoprotein covalent bond can be subsequently disrupted to elute the protein of interest with a pH change or by competitive
                elution using a sugar such as sorbitol.
 (pdf)
        QIAGEN:  
Qproteome Glycoprotein Fractionation Handbook. For the fractionation of glycoproteins in proteomic samples  (pdf)
              Qproteome Total Glycoprotein Kit. The Total Glycoprotein Spin Columns in the Qproteome Total Glycoprotein Kit contain ConA
               and WGA lectins. They are used for a general enrichment of the total glycoprotein population from a cell or serum sample.
               Qproteome Mannose Glycoprotein Kit. The ConA, GNA, and LCH lectin spin columns in the Qproteome Mannose Glycoprotein
               Kit are used for specific enrichment of glycoproteins with mannose-rich glycan moieties. The three lectins each bind different
               subclasses of these moieties.
               Qproteome Sialic Glycoprotein Kit. The WGA, SNA, and MAL lectin spin columns in the Qproteome Sialic Glycoprotein Kit
               are used for specific enrichment of glycoproteins with sialicacid-rich glycan moieties. The three lectins each bind different
               subclasses of these moieties.
               Qproteome O-Glycan Glycoprotein Kit. The AIL, and PNA lectin spin columns in the Qproteome O-Glycan Glycoprotein Kit are
               used for specific enrichment of glycoproteins with a glycan structure of the type that are found on T-antigens. The two lectins each
               bind different subclasses of these glycoproteins.

        QIAGEN:  Qproteome GlycoArrays is a simple, rapid, kit-based method for determining the pattern and relative abundance of specific
                  mammalian glycosylation epitopes in a glycosylated protein. The analysis can be performed on crude samples in growth media,
                  eliminating the need for time-consuming purification and sample preparation steps. The technology consists of arrays of selected lectins,
                  which are used to determine the glycosylation features of the analyzed glycoproteins.
 (pdf)  

Mimetics Ligands, Antibody fragments and Similar Technology

        BAC (BioAffinity Company)  CaptureSelect® Products. BAC BV provides unique products for affinity purification. These CaptureSelect®
                products are created by BAC’s proprietary technology based on Camelid derived single domain antibody fragments. These antibody
                fragments are produced efficiently by our host-vector system based on the yeast Saccharomyces cerevisiae, enabling high level production
                with minimal purification effort as well as easy scale-up. CaptureSelect® products possess a combination of unique properties such as
                stability, affinity, and selectivity that provides competitive benefits in terms of reduced cost of purification, higher quality product, and
                increased flexibility in the purification process. 
                Custom ligands.  CaptureSelect® technology enables new opportunities in the field of custom designed ligands. BAC has developed an
                unique ligand discovery program that is applicable to a wide variety of target molecules, ranging from bacteria and viruses to proteins,
                carbohydrates and even small molecules like haptens and peptide tags. (pdf-1)   (pdf-2)   (pdf-3)
            PROMETIC   Mimetic Ligand™ Affinity Chromatography. The Mimetic Ligand adsorbent range has been designed to enable
                purification of many proteins, providing a general technique that can replace ion-exchange, gel permeation and hydrophobic interaction
                chromatography, but with higher recovery and purity. Mimetic Ligand Screening Column Kit contains 1ml pre-packed columns of ten different
                Mimetic LigandTM adsorbents, suitable for attachment to automated chromatography work stations.
 (pdf)
            
SCIL Proteins      Affilin™ molecules are small non-immunoglobulin proteins which are designed for specific binding of proteins and small
                molecules. New Affilin™ molecules can be quickly selected from libraries which are based on two different human derived scaffold proteins.
                Affilin™ molecules can be further modified for various applications. Additional functionalities can be generated through genetic fusions with
                enzymes or effector domains.                     
                Specific labeling, chemically or with isotopes, extends the field of applications for Scil Proteins individualised binding proteins. 
                Affilin™ proteins are easily produced in the cytoplasm of E. coli and are fully functional without further posttranslational modifications such as 
                glycosylation. Affilin™ molecules will find their applications in therapy, chromatography, in vivo and in vitro diagnostics and as research tools. 

Albumin and Other Abundant Serum Proteins Removal        

      AGILENT   The Agilent Multiple Affinity Removal System for Proteomics Sample Preparation. Designed to remove greater than 98-99%
                of six interfering high-abundant proteins using Affinity-purified polyclonal antibodies to: albumin, IgG, IgA, transferrin, haptoglobin,
                and antitrypsin from human serum or three proteins (albumin, IgG, and transferrin) from mouse serum samples
 (pdf-I)   (pdf-II)
                (pdf-III)  (pdf-IV)  (pdf-V)
         AGILENT
   The Agilent Multiple Affinity Removal System. Designed to remove six interfering high-abundant proteins from Monkey
              Plasma for Proteomics Sample Preparation
 (pdf)
      
AGILENT   Agilent Human 14 Multiple Affinity Removal System Columns for the Fractionation of High-Abundant Proteins from Human
                Proteomic Samples  (pdf)
      GE-HEALTHCARE    Capto™ Blue a new affinity chromatography prototype resin for purification or removal of HSA.Made by
                immobilizing Cibacron™ Blue to a new High Flow Agarose matrix  (pdf)
      CALBIOCHEM   ProteoExtract™ Removal Kits (for Enhancing Resolution of Low Abundance Proteins)  (pdf)
         NORGEN   ProteoSpin™ Abundant Serum Protein Depletion Kit. The kit depletes 70% of albumin, 90% of α-antitrypsin, and 50% of transferin
                 and haptoglobin, enabling the visualization of low abundance proteins. The kit is unique in that it is based on an ion-exchange mechanism, silicon
                 carbide (SiC), and not the use of specific antibodies. As a result, the kit can be used to deplete serum proteins from a wide variety of samples,
                 including human and various animals.
(pdf)
        PIERCE:     SwellGel® Blue Albumin Removal Kit  (pdf)  
        QIAGEN:   Qproteome Albumin/IgG Depletion Handbook. Fast and specific removal of albumin and IgG from human serum and plasma samples.
                     Based on monoclonal antibodies that bind HSA and human IgG with high affinity and specificity.
  (pdf)
        SIGMA:      ProteoPrep 20 Plasma Immunodepletion Kit is a complete kit with all necessary reagents and consumable equipment to deplete 20
               high abundance proteins from human plasma or serum. This kit is designed to specifically remove the 20 proteins from human plasma.
               Specifically, 8 mL of plasma may be depleted in preparation for proteomic analysis, two-dimensional electrophoresis (2DE), or
               liquid chromatography (LC).
 (pdf).  

         VIVASCIENCE:  Vivapure® Anti-HSA Kit for Human Albumin Depletion 
                Highly specific human albumin depletion with unique antibody fragments  (pdf-I)  (pdf-II)
 (pdf-IV)   (pdf-V)
                Vivapure Anti-HSA Affinity Resin
 (pdf-III)    
         VIVASCIENCE:Vivapure® Anti-HSA/IgG Kits for Human Albumin and Human Albumin/IgG Depletion  (pdf)

   Chromatofusing
    
      
GE-HEALTHCARE    Chromatofusing Handbook   (pdf)
      
GE-HEALTHCARE    Ion Exchange Cromatography and Chromatofusing Handbook(pdf)

Crystallography and Recombinant Methods

    HAMPTON:  Solubility & Stability Screen is designed to assist in the identification of solution conditions which promote protein solubility and stability,
            and minimize
protein precipitation. Solubility & Stability Screen is a solubility screen, a stability screen, and may also be used as an additive screen
            in the presence
of a crystallization reagent.   (pdf-I)   (pdf-II)
     
JENA Macromolecular crystallography products. Crystallization Screens: JBScreen Basic and JBScreen Membrane. Crystallization Optimization:
            JBS Solubility Kit, JBS Methylation Kit, JBScreen Plus, JBScreen Detergents, JBScreen Buffer Kits and JBScreen Cryo  (pdf-I)   (pdf-II)

     
JENA   Crystallization Freshman Kit - Junior and Crystallization Freshman Kit - Scholar are designed for screening of initial crystallization
           conditions of  proteins, peptides, nucleic acids and macromolecular complexes in order to grow single crystals suitable for X-ray diffraction
           analysis. They include everything you need to start your crystallization experiment: pregreased sitting drop plates, cover slides, our unique
           JBScreen reagents as well a detailed user guide.  (pdf-I)  (pdf-II)  (pdf-III)  (pdf-IV)  (pdf-V)
     JENA   Protein Crystallization Starter Kit. Introduction and Theory of Crystallization. Which Materials are Useful as Precipitants   (pdf)    

     Derewenda Z.,  The use of recombinant methods and molecular engineering in protein crystallization.
         Methods (2004), 34: 354–363   (pdf)  
     Smyth D.,
 REVIEW  Crystal structures of fusion proteins with large-affinity tags.
        Protein Science (2003), 12:1313–1322
 (pdf) 
   

     Geerlof A. et.al.     The impact of protein characterization in structural proteomics.  
        Acta Cryst.(2006) D62: 1125-1136  
(pdf) 

     Newby Z. et.al.  A general protocol for the crystallization of membrane proteins for X-ray structural investigation.
        Nature Protocols (2009) 4: 619-637  (pdf)  
     Lee J. et.al.
 An efficient platform for screening expression and crystallization of glycoproteins produced in human cells.  
        Nature Protocols (2009) 4: 592-604  
(pdf)

Deglycosylation

      NORTHSTAR   Enzyme Deglycosylation Kit. Contains all enzymes needed to completely remove all N- & simple O-linked carbohydrates
            from glycoproteins
 (pdf)
      QAbio    Enzymatic CarboRelease Kit will remove all N-linked oligosaccharides and many O-linked oligosaccharides from glycoproteins.
            N-links (Asn-linked) are removed using the enzyme PNGase F. In addition, all Ser/Thr-linked (O-linked) Gal-(β1-3)-GalNAc-(α1) and
            all sialic acid substituted Gal-(β1-3)-GalNAc-(α1) will be removed using the combination of Sialidase and O-Glycosidase. The addition
            of ß-Galactosidase and Hexosaminidase will assist in the deglycosylation of larger O-link structures.  (pdf)
      PROZYME-GLYKO     Enzymatic Deglycosylation Kit. Contains all enzymes & reagents needed to completely remove all N-linked & simple
              O-linked carbohydrates from glycoproteins
 (pdf)
      ROCHE   The N-Glycosidase F Deglycosylation Kit can be used to test for the existence of asparagine-linked glycan chains on glycoproteins.
             The new Endoglycosidase H Deglycosylation Kit is useful for testing the existence of “high mannose” type and “hybrid” type asparagine-linked
              glycan chains on purified glycoproteins.  (pdf-I)    (pdf-II)
        SIGMA    
Deglycosylation Selection Guide   (pdf)

Expanded Bed Adsorption
 
      
GE-HEALTHCARE   Introduction to Expanded Bed Adsorption  (pdf)
      
GE-HEALTHCARE   Streamline Application Note  (pdf)   Streamline Data File   (pdf)   
      BIORAD  CHT Ceramic Hydroxyapatite: Use in Expanded Bed Adsorption Mode  (pdf)

     

Gel Filtration

    Protein concentration by ultrafiltration

      GE-HEALTHCARE   Gel Filtration Handbook   (pdf)  
      
GE-HEALTHCARE   Gel Filtration of peptides - buffer conditions  (pdf)  
     
 GE-HEALTHCARE  Packing of Gel Filtration column  (movie)   
      
GE-HEALTHCARE  Column evaluation (movie) 
      GE-HEALTHCARE   Superdex 200 Increase column  (pdf)
      MERCK Fractogel® EMD BioSEC (S) (pdf) 
      
SEPAX  Zenix SEC; silica 3µm particle size analytical column  (pdf)
      SEPAX  SEC-C line columns: for samples that shows a tendency to stick to traditional size exclusion resins with delayed elution time,
            low recovery, varying HMWS, or excessive tailing  (pdf-user manual)   (pdf-comparision)
      
TOSOH   TSKgel SW series SEC columns. TSKgel SW, SWXL and SuperSW columns are stable from pH 2.0 to 7.5 and have
            excellent solvent stability up to 100% organic solvent.  (pdf)   
      
TOSOH   Chromatographic Media Catalog   (pdf)
  
    TOSOH   Size Exclusion Chromatography (pdf-I)  (pdf-II)  (pdf-III)  (pdf-IV)

Hydrophobic Interaction Chromatography
          
      BioToolomics:  SepFast Butyl, SepFast Pentyl, SepFast Hexyl, SepFast Phenyl, SepFast Heptyl and SepFast Octyl are hydrophobic
            interaction chromatography (HIC) adsorbents having a carbon chain of C4,
C5, C6, C6 ring, C7 and C8, respectively. There is a
            choice of 3 different base matrices according to the pore accessibility of target molecules: 400 serial) to purify peptides or small
            proteins;
500 serial) to purify most medium to large proteins, and  600 serial) to purify large to very large proteins.  (pdf)
      GE-HEALTHCARE  Butyl-S Sepharose™ 6 Fast Flo  (pdf)
       
GE-HEALTHCARE  Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods  (pdf)
       
GE-HEALTHCARE  Hydrophobic Interaction Chromatography Manual  (pdf)   
       
GE-HEALTHCARE  HiTrap™ HIC Selection Kit consists of six Hydrophobic Interaction Chromatography media (HIC) with different
            hydrophobic characteristics. The kit provides the possibility to screen for the most appropriate HIC medium for specific applications.
            (pdf-I)  (pdf-II)
      
GE-HEALTHCARE   RESOURCE™ ETH (ether), ISO (isopropyl) and PHE (phenyl) are pre-packed high performance columns for
             separating biomolecules by hydrophobic interaction chromatography (HIC)   (pdf)
      
GE-HEALTHCARE  Packing of IEX, Affinity or HIC column  (movie)
      
GE-HEALTHCARE  Column evaluation (movie) 
      BIORAD  Macro-Prep Hydrophobic Interaction Chromatography Support - Instruction Manual  (pdf-I)   (pdf-II)
      MERCK  Cleaning and Regeneraton of  Fractogel EMD sorbents   (pdf)
      MERCK  Fractogel EMD
Phenyl (S) Hydrophobic Interaction Chromatography (HIC)   (pdf)
      MERCK  Fractogel EMD
Propyl (S) Hydrophobic Interaction Chromatography(HIC)  (pdf)

      TOSOH   Use of Hydrophobic Interaction Chromatography With a Non-Salt Buffer System for Improving Process Economics in Purification
                of Monoclonal Antibodies (pdf)      
     
 TOSOH   Chromatographic Media Catalog   (pdf)
      TOSOH   HIC Effects of Mixed Electrolites on Protein Separation (pdf)
      
TOSOH   Hydrophobic Interaction Chromatography   (pdf_I)    (pdf-II)

Hydroxyapatite

       BIORAD  Bio-Gel® HT - Bio-Gel HTP DNA Grade Bio - Gel HTP Hydroxyapatite - Instruction Manual  (pdf)
       BIORAD CHT Ceramic Hydroxyapatite  (pdf)
       BIORAD  CHT Ceramic Hydroxyapatite: Use in Expanded Bed Adsorption Mode  (pdf) 
       BIORAD  CFT Ceramic Fluoroapatite - A Chromatographic Support  for Protein Purifications Requiring Acidic Conditions  (pdf)
       
BIO RAD   Protein A removal  from IgG on CHT Ceramic Hydroxyapatite Support   (pdf)
       BIORAD   Macro Prep  Chromatography Supports   (pdf)
       Pall   HA Ultrogel Hydroxyapatite Chromatography Sorbents (pdf)

Ion Exchange Chromatography
 
      
BioToolomics:  SepFast HighRes Q, SepFast HighRes DEAE, SepFast HighRes S and SepFast HighRes CM are, respectively, 
                strong anion, weak anion, strong cation and weak cation exchange adsorbents. They
are specially designed for high resolution 
                purification of biological molecules. For each type of ion exchange medium, there is a choice of three different base matrices according
                to pore accessibility of target molecules  (pdf)

     
BioToolomics:   SepFast Purifier Q, SepFast Purifier DEAE, SepFast Purifier S and SepFast Purifier CM are, respectively, strong anion, 
                weak anion, strong cation and weak cation exchange adsorbents. They
are specially designed for large-scale purification of biological 
                molecules. 
For each type of ion exchange medium, there is a choice of three different base matrices according to pore accessibility of 
                target molecules  (pdf)

      
BioToolomics:   SepFast Capture Q, SepFast Capture DEAE, SepFast Capture S and SepFast Capture CM are, respectively, 
                strong anion, weak anion, strong cation and weak cation exchange adsorbents. They
are specially designed for cost-effective large-scale 
                capturing of biological molecules in the initial
downstream recovery step. 
For each type of ion exchange medium, there is a choice of three 
                different base matrices according to pore accessibility of target molecules  (pdf)

      
BioToolomics:  SepFast Supor Q a strong anion exchange chromatography product. The working medium possesses a combination of small 
                pore (50-100nm) and large pore (micro level). It shows fast accessibility to both small and large molecules (such as endotoxin, DNA,
                virus and virus like particles)   (pdf)
      CryoBioPhysica   pISep buffers
for separation of proteins by anion or cation exchange chromatography, applying user control external pH
                gradients. Can be apply to all kind of ion exchange columns, together with NaCl and Urea gradients.
pISep software to calculate pH
                gradients in the presence of salt, urea and some non-ionic detergents. Introduction (pdf) and overview of technology (pdf)

      GE-HEALTHCARE   Ion Exchange Chromatography Manual   (pdf)  
        
GE-HEALTHCARE    Ion Exchange Cromatography and Chromatofusing Handbook (pdf)   
        
GE-HEALTHCARE    Ion Exchange Media - Selection Guide   (pdf)    (pdf-II)
        
GE-HEALTHCARE  Recommended buffers for Anion Exchange Chromatography
        
GE-HEALTHCARE  Recommended buffers for Cation Exchange Chromatography
        
GE-HEALTHCARE  Recommended Volatile buffers systems for Ion Exchange Chromatography
        
GE-HEALTHCARE   Capto adhere: a strong anion exchanger with multimodal functionality ionic interaction, hydrogen bonding and
                  hydrophobic interaction. Capto adhere is designed for post Protein A purification of monoclonal antibodies. Removal of leached
                  Protein A, aggregates, host cell proteins, nucleic acids and viruses from monoclonal antibodies is performed in flowthrough mode
                  at where the antibodies pass directly through the column while the contaminants are adsorbed.  (pdf-I)  (pdf-II)  (pdf-III)
        GE-HEALTHCARE   Capto™ MMC: a multimodal cation exchanger. Has a multimodal ligand that may interact with target molecules
                  in several different ways. It contains a carboxylic group and thus its features partly resemble those of a weak cation exchanger.
                  In addition to the ionic interactions several other types of interactions are involved, including hydrogen bonding and hydrophobic
                  interaction.
The design of the ligand enables binding of proteins at high conductivity.  (pdf-I)  (pdf-II)  (pdf-III) 
        
GE-HEALTHCARE   MacroCap™ SP is a highly porous cation exchanger high available surface area for adsorption of of large
                  biomolecules  such as IgM and polyethylene glycol(PEG)-modified proteins (i.e., PEGylated proteins) that are intended for use as
                  biopharmaceuticals.  (pdf)
        
GE-HEALTHCARE   Q-Sepharose XL (anion exchange). Q-Sepharose XL virus licensed. SP-Sepharose XL (cation exchange) (pdf)   
        
GE-HEALTHCARE   Capto™ Q a strong anion exchange medium for packed bed chromatography that allows increased speed and
                 throughput in capture and intermediate purification. It combines high capacity with high flow velocity and low backpressure
 (pdf)
       
GE-HEALTHCARE   ANX Sepharose™ 4 Fast Flow (high sub) is a weak anion exchanger with different selectivity. Larger pores than
                DEAE Sepharose Fast Flow which improves the dynamic binding capacity when separating larger molecules  (pdf)    
        
GE-HEALTHCARE  Packing of IEX, Affinity or HIC column  (movie)
        
GE-HEALTHCARE  Column evaluation (movie)
        BIORAD  Macro-Prep Ion Exchange Support - Instruction Manual  (pdf)
        BIORAD UNOTMQ&S Continuous   (pdf)  (pdf  II)
        MERCK  Cleaning and Regeneraton of   Fractogel EMD sorbents   (pdf)
        MERCK Ion Exchange Chromatography Using Fractogel EMD Tentacle Supports  (pdf)   
        MERCK Fractoprep® DEAE Weak Anion Exchange chromatography   (pdf)
        MERCK Fractoprep® SO3 - Strong Cation Exchange chromatography   (pdf)
        MERCK Fractoprep® TMAE  Strong Anion Exchange (pdf)
        MERCK Fractogel EMD COO- (S) (M) Weak Cation Exchange
 (pdf)
        MERCK Fractogel EMD DEAE (S) (M) Weak Anion Exchange
 (pdf)
        MERCK Fractogel EMD DMAE (S) (M) Weak Anion Exchange
 (pdf)
        MERCK Fractogel EMD SE High Capacity (M) Strong Cation Exchange  (pdf)
        MERCK Fractogel EMD SO3- (S) (M) Strong Cation Exchange
 (pdf)
          MERCK Fractogel EMD TMAE (S) (M) Strong Anion Exchange  (pdf)
        MERCK Fractogel EMD TMAE High Capacity (M) Strong Anion Exchange
 (pdf)   

       MERCK Eshmuno™ S is a cation exchanger specifically designed for highly productive downstream purification of monoclonal antibodies. 
                 Hydrophilic polyvinyl ether base matrix that allows the use of much higher flow rates.  (pdf)    
       NATRIX  NatriFlo HD-Q Membrane is an advanced material with a three-dimensional macroporous hydrogel structure that provides a High
                 Density of binding sites and rapid mass transfer.  NatriFlo HD-Q Membranes deliver binding capacity that exceeds resin-based columns
                 with fast flow-rates typical of membrane adsorbers. (pdf  Instruction Guide)   (pdf  Method development)  (pdf quick start guide)
      PIERCE
  Pierce Strong Ion Exchange Spin Columns; use membrane-adsorbent technology as a chromatographic matrix to fractionate proteins.
                 The spin column capacities are 4 mg proteins/peptides for the mini and 60-80 mg protein for the maxi. Actual capacity depends on
                  the specific protein sample, selected pH and salt condition.  (pdf)
      
Pall    Q, S, DEAE, CM Ceramic HyperD ion exchangers. Of particular value in process scale application, these sorbents are designed to
                 maintain high dynamic binding capacity (DBC) under conditions where conventional sorbents display significant capacity or productivity
                 limitations.  (pdf)   Prepacked RPC columns (pdf)

       Pall   Pall® PRC Columns Prepacked Columns for Ion Exchange and Mixed-Mode Chromatography  (pdf)
      
Pall    Purification of  mouse IgM from cell culture supernatant by Cation Exchange Chromatography on CM Ceramic HyperD F sorbent  (pdf)
       
Pall    Q, S HyperCel™ ion exchangers. Different selectivities with improved productivity, available in prepacked PRC 1mL and 5mL column and 
                 in bulk. (pdf)   Article describing comparative characteristics and advantages. (pdf-II)
       Pall    Q, S Mustang® Membrane Adsorbers. These single-use or reusable capsules can be used for small scale quick purification at lab
                scale with  XT Acrodisc®, or even scaled-up. Capacities are several times higher than traditional resins, especially for large molecules (DNA
                or viruses). No packing is needed and it is compatible with existing pumping systems, or can be used with a syringe.  (pdf)
 
      SARTORIUS   SartobindTM - Membrane Adsorbers Discs and Cassettes. Reusable Sartobind Membrane Ion Exchangers for Adjustable
                Filter Holders  (pdf) 
       SARTORIUS   Sartobind® Membrane Adsorbers - A Separation Technology Based on Microporous Membrane Ion Exchangers  (pdf)
       SARTORIUS   Sartobind SingleSep Disposable Capsules - A Separation Technology Based on Microporous Membranes -
                Operating Instructions (pdf)
      
TOSOH    TSK-GEL BioAssyst Series Ion Exchange Column  (pdf)
       TOSOH     Ion Exchange Chromatography  (pdf)  
     
 TOSOH   Chromatographic Media Catalog   (pdf)
       VIVASCIENCE:   Vivapure™ IEX kits include everything required for rapid purification of protein samples or contaminant removal from
                  protein samples: clarification spin columns for initial sample clearing, Vivapure spin columns and ready-to-use buffers in different
                  concentrations for the protein bind-wash-elute steps, and Vivaspin™ ultrafiltration devices for final sample concentration and desalting.
                  Basic and acidic protein purification kits.
 (pdf)

Multimodal and Hydrophobic Charge-Induction Chromatography 

         BioToolomics:  SepFast MM AH-1 a mixed mode chromatography medium. The ligand contains a combination of anionic and
                hydrophobic groups. 
The product shows good binding capacity to molecules rich of hydrophobic moieties. It shows little binding
                to DNA or albumins under moderate
 ionic strength (e.g. 0.15 M salt)  (pdf)
       
GE-HEALTHCARE   Multimodal Chromatography Handbook (pdf)
        GE-HEALTHCARE   Capto adhere: a strong anion exchanger with multimodal functionality ionic interaction, hydrogen bonding and
                 hydrophobic interaction. Capto adhere is designed for post Protein A purification of monoclonal antibodies. Removal of leached
                Protein A, aggregates, host cell proteins, nucleic acids and viruses from monoclonal antibodies is performed in flowthrough mode
                at where the antibodies pass directly through the column while the contaminants are adsorbed.  (pdf-I)  (pdf-II)  (pdf-III)
        GE-HEALTHCARE   Capto™ MMC: a multimodal cation exchanger. Has a multimodal ligand that may interact with target molecules
                in several different ways. It contains a carboxylic group and thus its features partly resemble those of a weak cation exchanger.
               In addition to the ionic interactions several other types of interactions are involved, including hydrogen bonding and hydrophobic
                interaction.
The design of the ligand enables binding of proteins at high conductivity.  (pdf-I)  (pdf-II)  (pdf-III)  
        
GE-HEALTHCARE    Capto core 700, a media for flow through applications in intermediate purification and polishing of viruses and
                other large biomolecules. By using Capto Core 700 it is possible to remove contaminants Mr <~700kDa and allow for larger targets
                to be collected in the flowthrough. Capto Core 700 is targeted for flow through applications in intermediate purification of viruses and
                other large biomolecules. The novel core bead technology allows each bead in this medium to be designed with a ligand-activated core
                and an inactivate shell (a shell without ligands). The inactive shell excludes large biomolecules from entering the core through the pores
                in the shell. These larger biomolecules are therefore collected in the flowthrough while smaller contaminants enter through the pores in
                the shell and bind to the internalized ligands. The use of Gel filtration chromatography is a bottleneck in vaccine processes (polishing
                steps) because of the low column loading and slow flow rates. Capto Core 700 offer a high productivity solution to size exclusion
                chromatography in this field.  (pdf-data file)  (pdf-instructions)  (pdf-Influenza purific)

       Pall   MEP HyperCel was the first modern Mixed-Mode media on the market, combining hydrophobic interaction for binding and ionic
              repulsion for eluting. It can help purify antibodies and fragments as well as recombinant proteins (enzymes and others) with less or
              even no salt compared to HIC, while allowing an anionic repulsion pH drop at low conductivity. Together with HEA and PPA
              chemistries, these 3 ligands can be screened at the same time since providing different selectivities.
  (pdf)
       
Pall   Pall HEA and PPA HyperCel operate on a "mixed-mode" mechanism, based on a combination of electrostatic and hydrophobic
               properties of the protein and ligands. Ligands: aliphatic (HEA – hexylamine) and aromatic (PPA – phenylpropylamine) amines,
               which offer different selectivity and hydrophobicity options  (pdf)
               
Poster on different selectivities  (pdf)
       Pall   Pall HEA and PPA HyperCel operate on a "mixed-mode" mechanism, based on a combination of electrostatic and hydrophobic
                properties of the protein and ligands. Ligands: aliphatic (HEA – hexylamine) and aromatic (PPA – phenylpropylamine) amines,
                which offer different selectivity and hydrophobicity options. (pdf)
       
Pall   Purification of mIgG1 on MEP HyperCel Mixed-Mode media. Optimization  (pdf)
       
Pall   Pall® PRC Columns Prepacked Columns for Ion Exchange and Mixed-Mode Chromatography  (pdf)
       TOSOH   TOYOPEARL MX-Trp-650M: a new multimodal cation exchange resin for protein purification. It combines
                the selectivity options of mixed-mode chromatography with the binding capacity of modern ion exchange resins. Excellent choice for
                intermediate and polishing steps, such as aggregate removal in antibody purification.  (pdf)

        TOSOH   Chromatographic Media Catalog   (pdf)

Phospho-Protein Purification

       CLONTECH:  BD Phosphoprotein Kit User Manual (pdf) (pdf-II)   (pdf-III)      
       QIAGEN  Phosphoprotein Purification Kit. For purification and analysis of phosphorylated proteins from eukaryotic cells
     (pdf-I)   (pdf-II)

       Phos-Tag   Zinc (II) Phos-tag beads for enrichment of phosphorylated proteins from cell lysate using phosphate-affinity chromatography at 
             physiological pH  (pdf)  (pdf-I)  (pdf-II)  (pdf-III)  
       PIERCE:   Thermo Scientific Pierce Fe-NTA Phosphopeptide Enrichment Kit for efficient enrichment of phosphorylated peptides by spin 
            columns. Useful for Sample Prep for Mass Spec Analysis.  (pdf)

Polyubiquitin-modified proteins

        PIERCE   Ubiquitin Enrichment Kit. For the isolation and study of intracellular polyubiquitin-modified proteins. Through the use of a
              high-binding affinity resin, polyubiquitinated proteins are isolated from cell or tissue lysates. The bound proteins can then be eluted
              from the affinity resin and analyzed using the anti-ubiquitin antibody.
 (pdf)

Protein Aggregation

     Additives Used to Stabilize Folding and Prevent Aggregation

     Buffer solubility screen to avoid aggregation during protein concentration and ultrafiltration

      
Lebendiker M., and Danieli T. (2014)  Production of prone-to-aggregate proteins (Review)
            FEBS Letters 588: 236–246
- DOI information: 10.1016/j.febslet.2013.10.044  (pdf)
      Pall  AVACTA   Optim 1000 developed to reduce the time and cost of therapeutic protein pre-formulation studies, stability testing and formulation.
           Thermal unfolding and aggregation curves are simultaneously acquired for 48 samples run in a single experiment, enabling 96 samples to
           be analysed in one working day. Sample volumes as low as 1 μl. Simultaneous optical measurements: 1) Intrinsic fluorescence to monitor
           tertiary structure  2) Static light scattering to detect aggregates. Sample heating and cooling to determine: 1) Protein unfolding temperature
           (Tm) 2) Protein aggregation onset temperature (Tagg) 3) Time dependent unfolding and aggregation at a fixed temperature   (pdf)
      BioTek  and ENZO ProteoStat® Protein aggregation assay provides a simple, homogenous assay format for monitoring protein aggregation in a
          microplate assay format. The assay can be employed to streamline protein processing and optimize formulation procedures; in a wide pH
          and ionic strength range. The Synergy™ Mx Multi-Mode Microplate Reader is used for all assays. Its quadruple monochromator system
          in top-reading mode is used with slit widths of 9 nm. The excitation monochromator is set to 500 nm and emission to 600 nm.
         (pdf-manual)  (pdf-I)  (pdf-II)
      DILYX Biotechnologies   OptiSol Protein Solubility Screening Kit Application Manual. Array-based filtration technology that enables to either:
           1) identify formulations that protect a target protein from aggregation, or 2) gently solubilize an aggregated protein  sample. This screening kit
           contains a systematically varied array of buffers (from pH 3 to pH 10) and a series of solubility enhancers (salts, amino acids, sugars, polyols,
           reducing reagents) that enable the determination of conditions under which a particular protein sample is protected from aggregation or can be
           de-aggregated. Filtration principle: soluble proteins pass the filter due to their smaller size, while aggregated proteins do not pass filter.  (pdf_I)
            (pdf II)
    
   HAMPTON:  Solubility & Stability Screen is designed to assist in the identification of solution conditions which promote protein solubility and stability,
            and minimize
protein precipitation. Solubility & Stability Screen is a solubility screen, a stability screen, and may also be used as an additive screen
            in the presence
of a crystallization reagent.   (pdf-I)   (pdf-II)

      INTEGRITY BIOSOLUTION   Protein Aggregation  (page)    
      NOVAGEN  Enhancing solubility during protein expression in E. Coli  (site)    (site-II)
        PROTEIN EXPRESSION FACILITY OF THE HEBREW UNIVERSITY OF JERUSALEM  Heat shock growth procedure

      Bondos S.,  Detection and prevention of protein aggregation before, during, and after purification.                                                                                    
        Analytical Biochemistry
  (2003),
316 (2), 223-231  (pdf)
      Cromwell M.,  Protein Aggregation and Bioprocessing
        The AAPS Journal 2006; 8 (3) Article 66 pg.E-572  
(pdf)
      Hamada H., Effect of additives on Protein Aggregation   
        Current Pharmac. Biotech. (2009), 10, 400-407 (pdf) 
      Philo J.,   Mechanisms of Protein Aggregation
        Current Pharmac. Biotech. (2009), 10, 348-351  (pdf)                                                                             
     
Eiler S., Overexpression, Purification and Crystal Structure ….                                                                    
        Protein Expression and Purification (2001) 22, 165- 173
(pdf)


Protein Labeling with Biotin

       MOLECULAR PROBES    Biotin-XX Microscale Protein Labeling Kit.  This kit has been optimized for labeling  small amounts
               (20–100 μg) of purified proteins with molecular weights between 12 and 150 kDa, and contains everything needed
               to perform three labeling reactions and to separate the resulting conjugates from excess reactive biotin.(pdf)
   
       MOLECULAR PROBES    FluoReporter ®Biotin Quantitation Assay Kit for biotinylated proteins. Sensitive fluorometric assay for
              accurately determining the number of biotin labels on a protein. The assay can detect from 4 to 80 pmol of biotin in a sample.
 (pdf)

Protein Refolding / Inclusion Bodies
     
       
GE-HEALTHCARE   Purifying Challenging Proteins: Membrane proteins, Multiprotein complexes and Inclusion bodies  (pdf)
       AVIDIS-TECHNOLOGY Refolding Chromatography
            Refolding Chromatography with Mini-Chaperones (pdf)
       
AthenaES   QuickFold™ Protein Refolding Kit  with a Detergent and a Cyclodextrin Screening Kit   (pdf)
       BIO-VECTRA Vectrase AT - Folding Proteins with Disulfide Bands
       BIO-VECTRA Vectrase CD - Quick Screening of  Refolding Conditions (pdf)
       
BIO-VECTRA  Vectrase DK - Refolding at High Protein Concentration       
       BIO-VECTRA     Vectrase P - Protein Disulfide Isomerase (PDI) Mimic  (pdf-I)
(pdf-II)
       GENO-Protein Foldase-Protein Folding Optimization Kit
       NORGEN  The ProteoSpin™ Inclusion Body Isolation Kit. Facilitates the isolation of recombinant proteins in the form of inclusion bodies from E.
            coli. The kit includes reagents specially formulated to achieve rapid and high-quality purification of inclusion body proteins using three processes:
                1. Lysis of bacterial cells to release inclusion bodies in solid form
                2. Solubilization of purified inclusion bodies
                3. Purification of the recombinant protein using spin column chromatography with Norgen’s proprietary resin as an ion-exchanger
            Each spin column is able to purify up to 12 mg of recombinant proteins from 100 mL of culture. The kit is designed to purify both acidic and
            basic proteins.
 (pdf Maxi Kit)  (pdf Micro Kit)

      
NOVAGEN  Enhancing solubility during protein expression in E. Coli  (site)    (site-II)
       NOVAGEN-Protein Refolding kit (pdf)
       NOVAGEN Information on Protein Refolding (pdf)
       NOVAGEN  iFOLD™ Protein Refolding System . The system includes inclusion body purification reagents combined with a dispensed 96-well
            plate-based protein refolding buffer matrix.
 (pdf-I)    (pdf-II)

       NOVEXIN   The technology and reagents protect the protein during vulnerable steps in the refolding process, providing an opportunity for the
            protein to refold corrctly, reducing losses due to aggregation.
            Employs linear carbohydrate polymers of ~5kDa that enhance protein solubility and stability through the formation of reversible complexes with
            proteins without altering their structure and preventing aggregation.
            Protein Refolding  Starter Kit  (pdf)
            Stability P.A.C.  (pdf)
            Acidic Protein Refolding Kit  (pdf) 
         
  Basic Protein Refolding Kit  (pdf)  
            Refolding Kit for Histidine-tagged proteins  (pdf-I)   (pdf-II)
            Bulk Protection and Release Reagents  (pdf)

       PIERCE   Pro-MatrixTM Protein Refolding Kit (pdf)
       
       PROTEIN EXPRESSION FACILITY OF THE HEBREW UNIVERSITY OF JERUSALEM  Heat shock growth procedure
       PROTEIN EXPRESSION AND PURIFICATION FACILITY OF THE EUROPEAN MOLECULAR BIOLOGY LABORATORY 

        In vitro denaturation and refolding
      
Additives Used to Stabilize Folding and Prevent Aggregation
       Protein Refolding on IMAC resin - Batch Screening Procedure - On-Column Scale-up
       Contaminant Removal from Inclusion Bodies Before Solubilization

      UNIVERSITY OF OKLAHOMA  School of Chemical Engineering and Materials Science
 Recombinant Protein Solubility Prediction

       
    Recommended Reading

     Altamirano M., Refolding Chromatography with Immobilized Mini-Chaperones. PNAS 1997, 94: 3576-3578 (pdf)
       Armstrong N.,
A New Protein Folding Screen...etc. Protein Science 1999,  8: 1475-1483  (pdf)
       Chen G., Overexpression of a Glutamate Receptor (GluR2) ligand binding domain in E.Coli: Application of a novel protein folding screen   (pdf)
       De Bernardez Clark E.,Refolding of  RecombinantProteins  . Current Opinion in Biotechnology 1998,9:157–163  (pdf)
       De Bernardez Clark E., Protein refolding for industrial processes. Current Opinion in Biotechnology 2001, 12:202–207
(pdf).
       Eiler S., Overexpression, Purification, and Crystal Structure of Native ER alphaLBD  Protein Expression and Purification (2001) 22, 165–173  (pdf)
       Machida S.,
 Cycloamylose as an efficient artificial chaperone for protein refolding.  FEBS Letters 486 (2000) 131-135  (pdf)
       Middelberg A.,
Preparative Protein Folding. TRENDS in Biotechnology 2002, 20 (10): 437-443  (pdf)
       Ming Li et al., In vitro protein refolding by chromatographic procedures.  Protein Expr. and Purif. 2004,33: 1-10
 (pdf)
       Tsumoto K., Practical Considerations in Refolding Proteins from Inclusion Bodies. Protein Expr. and Purif. 2003,28: 1-8
 (pdf)       

  Reverse Phase Chromatography
        
GE-HEALTHCARE    Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods  (pdf)
        
GE-HEALTHCARE    Reverse Phase Chromatography Manual  (pdf)
        
PIERCE:   Peptide solubility guidelines. Use amino acid characteristics to predict hydrophobicity.  (pdf)
       
TOSOH BIOSCIENCE   Reverse Phase Chromatography Brochure  (pdf)  (pdf - new)
        VYDAC   The Handbook of Analysis and Purification of Peptides and Proteins by Reverse-Phase HPLC   (pdf)    

Simulated Moving Bed Chromatography

     SEMBA Biosciences:    Poster: Continuous highly efficient protein purification using simulated moving bed chromatography  (pdf)
     SEMBA Biosciences:    Application Note: The Semba Octave™ Chromatography System. Simulated moving bed chromatography.
            Continuous Affinity Purication of Recombinant Proteins  (pdf)  
     SEMBA Biosciences:    The Semba Octave™ Chromatography System. A simulated moving bed chromatography system. Continuous unattended
            purification, milligrams to grams. Compatible with chemical and biological samples.  SMBC emulates countercurrent separation where the mobile
           opposite direction of the stationary phase. The stationary phase is represented by individual columns connected in series, and the mobile phase by
            inlet streams of Feed and Desorbent and outlet streams of Raffinate and Extract. (pdf)  

Storage of Purified Proteins
    
       PROTEIN EXPRESSION AND PURIFICATION FACILITY OF THE EUROPEAN MOLECULAR BIOLOGY LABORATORY 
                  Storage of Purified Proteins
        PIERCE   Protein stability and storage  (pdf)

Thiophilic Chromatography
    CLONTECH: Thiophilic - Purification of Immunoglobulins (pdf) 
      CLONTECH:  Protein Purification Products (pdf)
      
GE-HEALTHCARE   Activated Thiol Sepharose 4B reacts with solutes containing thiol groups under mild conditions to form mixed disulphides.
            This reaction forms the basis of covalent chromatography and a procedure for immobilizing thiol containing biomolecules
.  (pdf)

      
GE-HEALTHCARE   Thiopropyl Sepharose  (pdf) 
      MERCK  Fractogel Thiophilic (pdf) 
      PIERCE:  T-Gel™ Purification Kit  (pdf)      
      MILLIPORE   PROSEP-Thiosorb and PROSEP-Thiosorb M Chromatography Media - for the recovery and purification of Immunoglobulins
 (pdf)

Viral Purification

    ADEMTECH    Viro-Adembeads are specifically developed magnetic particles to capture viruses from biological samples. Following
           incubation 
in a virus containing sample, Viro-Adembeads associated to viruses are recovered and can be directly used to infect target
           cells increasing
 infection efficiency. The mechanism of virus capture is based on electrosteric interactions
  (pdf-I)  (pdf-II)  
   
BIA Separations  CIM® Monolithic Columns  based on CIM Convective Interaction Media® Technology; suitable for purification of large biomolecules
            such as viruses (viral vectors and vaccines), DNA (plasmid DNA) and larger proteins (Immunoglobulins G and M, pegylated proteins).
            CIM
® Monolithic Columns exhibit unrivaled characteristics in terms of operational flow rates, binding capacity and separation
            resolution for large biomolecules.
Products are used in research, laboratory, pilot and industrial production stages and are extremely simple to use,
            with no packing of columns needed
    (pdf-I)  (pdf-II)  (pdf-III)  
    GE-HEALTHCARE   Q-Spharose XL (anion exchange). Q-Spharose XL virus licensed. SP-Spharose XL (cation exchange)  (pdf)
    
GE-HEALTHCARE   Rapid Adenovirus Purification Using Q-Spharose XL (pdf-I)  or Source 15Q (pdf-II)
    
GE-HEALTHCARE    AVB Sepharose™ High Performance is an affinity medium designed for the purification of adeno associated
            virus (AAV). The ligand, a 14 kD recombinant protein, is attached to the base matrix via a long, hydrophilic spacer arm to make it easily available
            for binding of the virus  (pdf)
   GE-HEALTHCARE    Capto core 700, a media for flow through applications in intermediate purification and polishing of viruses and
                other large biomolecules. By using Capto Core 700 it is possible to remove contaminants Mr <~700kDa and allow for larger targets
                to be collected in the flowthrough. Capto Core 700 is targeted for flow through applications in intermediate purification of viruses and
                other large biomolecules. The novel core bead technology allows each bead in this medium to be designed with a ligand-activated core
                and an inactivate shell (a shell without ligands). The inactive shell excludes large biomolecules from entering the core through the pores
                in the shell. These larger biomolecules are therefore collected in the flowthrough while smaller contaminants enter through the pores in
                the shell and bind to the internalized ligands. The use of Gel filtration chromatography is a bottleneck in vaccine processes (polishing
                steps) because of the low column loading and slow flow rates. Capto Core 700 offer a high productivity solution to size exclusion
                chromatography in this field.  (pdf-data file)  (pdf-instructions)  (pdf-Influenza purific)

    CLONTECH: Adeno-X™ Virus Purification Kits User Manual  (pdf-I)  Protocol (pdf-II). A complete filtration-based system for purifying and
               concentrating recombinant adenovirus. It provides a superior alternative to cesium chloride (CsCl) density gradient centrifugation. Use an
               adsorbent membrane that selectively binds adenoviral particles based on their distinctive surface-associated properties.
    CHISSO Corp   MILLIPORE  Matrex Cellufine Sulfate - For concentration, purification and depyrogenation of Virus, Viral/Microbial Antigen,
              Heparin binding proteins
 (pdf-I)    (pdf_II)   References: (pdf-III)
    Pall   Dynamic High Capacity Mustang® Q Membrane Units for Scaleable Anion Exchange Chromatography Purification of Adenoviral Vectors  (pdf)
    VIVASCIENCE:
 Vivapure® AdenoPACK™ 100. Adenovirus (Ad5) purification and concentration kit for up to 100 ml cell culture volume.
              AdenoPACK syringe filters containing an ion exchange membrane adsorber that binds adenoviral particles.
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Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem
mariol@cc.huji.ac.il  Tel: 972-2-6586920  

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