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

Protein Quantitation

Absorbance 280nm
BCA
Biuret
Bradford
CBQCA
Comparision of Methods
Coomasie
ESL
Biotinylated proteins
Glycoprotein Carbohydr.
Interfering Substances
Lowry
Nano Orange
Non-Interfering
OPA (fluorescent)
Preparation Reagent
Protein Precipitation


Gel Electrophoresis

 PAGE-SDS (Laemmli)
Basic-Native
Acidic-Native
Tricine 
Phosphate affinity SDS-PAGE
QPNC-PAGE

Protein extraction from Polyacrylamide Gel

Western-Blot


Proteomics Sample Preparation
Protein Precipitation 

Protein extraction from Polyacrylamide Gel 

Gel Stain
Coomasie Blue
Fluorescence
Glycoprotein
Lypopolysaccharide  

Oligohistidine
Phosphoprotein
Silver Stain
Zinc Stain




Proteases


Analytical 

Ultracentrifugation

Protein-Protein Interaction

Light Scattering

Circular Dichroism


Protein Quantitation   
  
Comparision of Different Protein Determination Methods
     Absorbance 280nm According toProtein Protocols in CD Rom
     Bradford     
     Lowry  According toProtein Protocols in CD Rom
     Lowry-Peterson  For membrane proteins, diluted solutions or solutions with interferants   
     Protein Precipitation Methods to concentrate or eliminate interferences before electrophoresis or protein determination

     ADVANSTA   Preparing Samples for Western Blot Analysis: Protein Quantification. A short explanation about different quantification methods  (pdf)
     BIO-RAD    Poster with a brief description of  protein assays from Bio-Rad, and reagent compatibility   (pdf) 
     BIO-RAD    Bradford Modified BIO RAD protein Assay  (pdf)
     BIO-RAD    Lowry Modified DC BIO-RAD (pdf)    (pdf-2)
     BIO-RAD    Lowry Modified RC DC BIO-RAD (pdf)   
     EXPEDEON  BradfordUltra™ kit is a quick and ready-to-use Coomassie-binding, colorimetric method for total protein quantitation in an environment 
            containing up to 1% detergent (1% high protein range, 0.1% low protein range). Is an improvement over classical Bradford formulations that cannot 
            tolerate detergent contamination of the protein samples.  (pdf)  
    GENO  Non-Interfering Protein Assay GENO Technology 
    INVITROGEN : Comparision of Different Protein Determination Methods
    INVITROGEN : CBQCA Protein Quantitation Kit   High-sensitivity protein quantitation (including lipoproteins and small peptides) even
            in the presence of lipids, detergents, reducing agents or membranes  .The CBQCA reagent becomes fluorescent upon reaction with amine groups
            on proteins (amine of lysine sidechains), so amines (e.g., Tris or glycine) and ammonium ions should be avoided.. Detects as little as 100 ng of
            protein/ml
    (pdf)

     INVITROGEN : Nano Orange  Range between 10 ng/mL and 10 µg/mL. The protein sample is  added to the diluted NanoOrange reagent, 
            mixture, heated at 95°C for ten minutes and fluorescence can be measured as soon as the mixture has cooled to room temperature. Tolerant to
            reducing agents and nucleic acids, not to detergents. Although unusually high concentrations of lipids in the sample can interfere with the assay, this
            interference can be eliminated by acetone precipitation of the protein, followed by delipidation with diethyl ether
   (pdf)
     INVITROGEN : Quant-iTTM Protein Assay Kit. Range between 0.25-5 µg of protein. The assay is performed at room temperature, and the
            signal is stable for 3 hours. Common contaminants, including salts, solvents, 2-mercaptoethanol, amino acids and DNA, are well tolerated in this assay.
            It is not compatible with detergents
  (pdf)     (pdf-II)
     
INVITROGEN :  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)
     
INVITROGEN : RediPlate EZQ Protein Quantitation Kit. Solid-phase, fluorescence-based protein assay that facilitates fast quantitation
            of protein samples prepared for gel electrophoresis. The assay can be performed in the presence of detergents, urea and reducing agents.
            Spot 1 µL of  protein sample onto the prepared paper, stain and read the fluorescence. Ideal for determining protein concentrations of samples
            prior to polyacrylamide gel electrophoresis. Effective range for the assay is 0.02–5 mg/mL, or 0.02–5 μg per spot
  (pdf)
     
INVITROGEN : EZQ Phosphoprotein Quantitation Kit (E33201) provides a fast and simple assay for phosphoproteins in solution.
            No radioactivity or antibodies are required, and sample analysis can typically be completed within 60 minutes. Compatible with samples
            containing detergents, reducing agents and urea buffers with up to 1% carrier ampholytes. Requires only 1 µL of sample, and up to 96 samples,
            including standards, can be assayed simultaneously. This kit is ideal for analyzing protein kinase or phosphatase activities, as well as for monitoring
            relative phosphoprotein concentrations during chromatography or after IEF fractionation of protein samples.  (pdf)
      
INVITROGEN : EZQ Phosphopeptide Quantitation Kit (E33202) permits accurate quantitation of phosphopeptides in solution, in the
            presence of standard buffer components. No radioactivity or antibodies are required, and sample analysis can typically be completed within 60 minutes.                   
            Requires only 1 µL of sample, and up to 96 samples, including standards, can be assayed simultaneously. This kit is ideal for analyzing phosphatase
            and kinase activities, as well as for monitoring relative phosphopeptide concentrations before and after analysis by liquid chromatography, mass

            spectrometry or other separation technique. Detection from 0.2–0.8 picomole to about 400 picomoles, depending on the peptide.
(pdf)
     OZ Biosciences 
Bradford Protein Assay Kit. The improved and optimized 1X Bradford reagent buffer allows superior linearity of response at
             low and high protein concentrations  (Low: 0.5 – 50 µg/mL High: 50 – 1500 µg/mL) and determination of protein amount in the presence
             of detergent (< 0.1%).  (pdf)
     
PIERCE  Protein Assay Technical Handbook  (pdf)   and   Protein Assay Compatibility Table
     PIERCE  Absorbance 280nm (pdf)
     PIERCE  
Bicinchoninic Acid KIT (BCA) PIERCE  Detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection
            and quantitation of total protein. Working range: 20–2,000 μg/ml. Color formation depends of: the macromolecular structure of protein, the number
            of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine).
            Certain substances are known to interfere with the BCA Assay including those with reducing potential, chelating agents, lipids and strong acids or
            bases. Interfering substances may be eliminated by dialysis or gel filtration, or diluting the sample until the substance no longer interferes, or
            precipitating the proteins in the sample with acetone or trichloroacetic acid (TCA)
  (pdf)
     PIERCE 
Bicinchoninic Acid KIT (Micro-BCA) PIERCE   (pdf)
     PIERCE 
Bicinchoninic Acid Method (BCA) PIERCE  The protein assay combines the well-known reduction of Cu+2 by protein to Cu+1 in an alkaline
            medium with the cuprous (Cu+1) ion detecting property of BCA.Color formation depends of: the macromolecular structure of protein, the number

            of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine). Reducing agents and copper chelators
            do interfere with this assay. Interfering substances may be eliminated by dialysis or gel filtration, or diluting the sample until the substance no longer
            interferes, or precipitating the proteins in the sample with acetone or trichloroacetic acid (TCA) 
  (pdf)
     PIERCE 
Bicinchoninic Acid Compatible Substances (BCA) PIERCE    (pdf)
     PIERCE 
BCA™ Protein Assay Kit−Reducing Agent Compatible. For quantitation of total protein in samples while minimizing interference
            from disulfide reducing agents. A compatibility reagent that modifies disulfide reducing agents is added to the sample before adding the
            BCA™ Reagents. This assay is also compatible with most ionic and non-ionic detergents in the presence of a disulfide reducing agent.
            Very useful for membrane proteins
. (pdf)
     PIERCE 
Biuret PIERCE   (pdf)
     PIERCE 
Coomasie Dye Binding KIT PIERCE  A modification of the well-known Bradford Coomassie® Dye-protein binding colorimetric method
            for total protein quantitation. Most ionic and nonionic detergents interfere with the assay. Interfering substances may be eliminated by dialysis or gel
            filtration, or diluting the sample until the substance no longer interferes, or precipitating the proteins in the sample with acetone or trichloroacetic acid
            (TCA)
   (pdf)
     PIERCE  Coomasie Dye Binding Method PIERCE   (pdf)   
     PIERCE  Glycoprotein Carbohydrate Estimation KIT PIERCE   (pdf)   
     PIERCE  Interfering substances PIERCE   (pdf)
     PIERCE   Lowry Method PIERCE    (pdf)   
     PIERCE  Lowry Modified KIT PIERCE (pdf)   
     PIERCE  o-Phthalaldehyde (OPA) Fluorescent Assay PIERCE (pdf)
     PIERCE    Protein Assay Preparation Reagent Compat-AbleTMPIERCE  (pdf)
     PIERCE   Protein Assay Interfering Substances PIERCE (pdf)
     ROCHE   ESL Protein Assay   (pdf)  
     SIGMA  Glycoproteomics Selection Guide — Labeling and Detection, Quantitation  (pdf)


Gel Electrophoresis
   PAGE-SDS (Laemmli)
      Basic-Native: For Acidic and Neutral proteins (pI < 7.0)
      Acidic-Native: For Basic proteins (pI > 7.0)
      Tricine: SDS gel for low MW proteins (<25 kDa) 
      QPNC-PAGE by Bernd Kastenholz: Quantitative Preparative Native Continuous Polyacrylamide Gel Electrophoresis (QPNC-PAGE). A highly
            efficient approach to resolve native and denatured metalloproteins (MW 6 to > 200 kDa) in complex protein mixtures. (pdf)  

      Phosphate affinity SDS-PAGE   Phosphate affinity polyacrylamide gel electrophoresis using Acrylamide-pendant Phos-Tag for the analysis of 
            phosphoprotein isotypes (pdf-I)  (pdf-II)  
            Separation and detection of large phosphoproteins by using Phos-tag SDS-PAGE. E. Kinoshita, et al., Nature Protocols, 4 (2009) pg.1513. (pdf) 

Western-blot

      ABCAM: Western blotting, a beginner's guide. Sample preparation. Electrophoresis. Transfer of proteins and staining. (pdf-I). Troubleshooting  (pdf-II)

Protein extraction from Polyacrylamide Gel
      GeBAflex-tube   Protein Extraction from Polyacrylamide Gel  (pdf)
      

Proteomics Sample Preparation
 
    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)
      ADVANSTA  Afyon SDS-PAGE sample preparation kit is a fast, efficient way to concentrate protein samples and remove buffer components that may
                    interfere with electrophoresis. In less than 10 minutes, samples are ready for SDS-PAGE or immunoblotting. The Afyon protocol is easily scaled up
                    and can be used for routine preparation of samples for electrophoresis.  (pdf-ApplicNote)  
 (pdf-Brochure)   (pdf-Short Protocol)  
                    
(pdf-User Manual)   (pdf-Protein Analysis)
    ADVANSTA   Preparing Samples for Western Blot Analysis: Protein Quantification. A short explanation about different quantification methods  (pdf)
    GE-HEALTHCARE  Protein Sample Preparation Handbook  (pdf)
                    Overview of protein sample preparation
                    Sample collection, stabilization, and protein extraction
                    Increasing detectability of targeted proteins
                    Ensuring compatibility in protein sample preparation workflows

   GE-HEALTHCARE 2-D Protein Extraction Buffer. Six buffers are available, supplied as a dry powder which is rehydrated with 
                    the provided diluent. The optimal buffer will depend on the nature of your sample, and 2-D Protein Extraction Buffer Trial Kit allows 
                    you to evaluate each extraction buffer to find the most suitable buffer for your sample. 2-D Protein Extraction Buffer can also be used 
                    prior to 1-D electrophoresis, or for rehydrating IPG strips prior to 2-D electrophoresis. (pdf)
    GE-HEALTHCARE  Mammalian Protein Extraction Buffer and Yeast Protein Extraction Buffer Kit are mild, detergent-based cell 
                    lysis methods to extract total soluble protein, eliminating the need for mechanical cell lysis, and delivering high quality protein lysate.  (pdf)
   
BIORAD  ProteoMiner™ Protein Enrichment Technology is based on treatment of complex protein samples with a large, highly diverse library
                    of hexapeptides bound to chromatographic supports. In theory, each unique hexapeptide binds to a unique protein sequence. Because the
                    bead capacity limits binding capacity, high-abundance proteins quickly saturate their ligands and excess protein is washed out during the
                    procedure. In contrast, low-abundance proteins are concentrated on their specific ligands, thereby decreasing the dynamic range of proteins
                    in the sample. ProteoMiner Protein Enrichment Kit utilizes single elution reagent. ProteoMiner Sequential Elution Kit utilizes multiple elution
                    reagents sequentially elute proteins based on different properties. (pdf-I)  (pdf-II)
       
    BIORAD  Strategies for Proteomics Sample Preparation  (pdf)
    CALBIOCHEM   ProteoExtract™ Removal Kits (for Enhancing Resolution of Low Abundance Proteins)  (pdf)      
   
GEBA  ProteoCon Kit Rapid and easy protein concentration by spin column. ProteoConD kit: use beads that are for concentration of protein in denatured
                    condition. ProteoConN kit: use beads that are for concentration of protein in native condition.  (pdf)

      GEBA   Maxi GeBAflex-tube Gel Extraction and Dialysis Kit (3 ml) Handbook. Applications: Extraction of proteins, RNA, DNA or oligonucleotides
                    (>20 nt) from polyacrylamide, agrose or any gel matrix in any running buffer; dialysis or buffer exchange of volumes between 0.1-3 ml; and
                    preparation of protein  samples for MALDI-MS. (pdf-I)  (pdf-II)

       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)

      NORGEN   ProteoSpin™ CBED (Concentration, Buffer Exchange and Desalting) Kit. The simultaneous removal of salts while concentrating a dilute
                    protein solution makes the kit a convenient method for preparing proteins before running many downstream applications such as SDS-PAGE
                    and isoelectric focusing. Purification is based on spin column chromatography using Norgen’s proprietary resin as the separation matrix. These
                    large columns are frequently used to prepare protein samples for structural analysis where larger amounts of protein are needed, such as
                    X-ray crystallography, NMR spectroscopy and other applications. The ProteoSpin™ CBED Maxi Kit comes with solutions for concentrating
                    and desalting both acidic and basic proteins.  (pdf for Micro)  (pdf for Maxi)

       NOVAGEN   A comprehensive selection of sample preparation tools for three main areas of protein research: expression proteomics, functional proteomics,
                    and structural proteomics   (pdf)

                    ProteoEnrich™ ATP-Binders™ Kit allows group separation of protein kinases and other ATP-binding proteins, yielding partially purified cell
                    extracts enriched in active protein kinases. (pdf-II)
                    ProteoExtract®Native Membrane Protein Extraction Kit is designed for the isolation of membrane proteins from mammalian cells and tissues.
                    The extremely mild procedure yields a solution of integral membrane and membrane-associated proteins in their non-denatured state. The
                    straightforward, two-step procedure does not require ultracentrifugation or incubation at elevated temperatures. (pdf-II)
                    ProteoExtract Phosphopeptide Capture Kit enables isolation of phosphorylated peptides derived from cleaved or digested protein samples or
                    kinase reactions designed for phosphorylation site identification.  
Combined with the ProteoExtract All-in-One Trypsin Digestion Kit, this kit has a
                    unique magnetic affinity resin to isolate phosphorylated peptides  with a high degree of specificity. 
(pdf-II)
                    BugBuster® Master Mix extracts active, soluble protein with maximum yield. It combines BugBuster Protein Extraction Reagent with Benzonase®
                    Nuclease and rLysozyme™ Solution in one convenient reagent. The all-in-one protein extraction reagent efficiently lyses bacteria and digests
                    nucleic acids.
                    ProteoExtract Abundant Protein Removal Kits. These kits facilitate highly specific depletion of either albumin or albumin/IgG from serum, plasma,
                    or cerebrospinal fluid. Selective removal of high-abundance protein improves the detection of low-abundance proteins of interest.  (pdf-II)
                    ProteoExtract® Protein Precipitation Kit. Efficient concentration and sample clean-up. Provides efficient concentration of proteins and removal
                    of interfering substances from dilute protein samples in a single step. The ProteoExtract Protein Precipitation Kit uses a unique combination of
                    precipitation agents to quantitatively precipitate proteins and remove interfering substances, for one-step concentration and clean-up of proteins
                    (pdf-II)

       NOVAGEN   Preparation of Protein sample for SDS-PAGE Procedures and Tips - Article (pdf)
       Pall   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)

        Pall       Enchant™ Albumin Depletion Kit. The albumin depleting discs utilize a Cibacron Blue based support that is re-hydrated to form a gel
                    based slurry. The gel based slurry is equivalent to 200 μL of resin. The easy five step protocol allows you to remove 2 mg of albumin from
                    each sample processed.
 (pdf)
        Pall       Enchant™ IgG Purification and Depletion Kits  (pdf)
        Pall       Enchant™ Multi-Protein Affinity Separation Kit includes all of the components necessary to fractionate albumin and IgG from 50 μL
                of normal human serum or plasma under native or denatured conditions.  (pdf)

       PIERCE:    Manual: Sample Preparation for 2-DE and Mass Spectrometry  (pdf)   
                    An Introduction to Mass Spectrometry.  Poppers™ Liquid Cell Lysis Reagents.  Mitochondria Isolation Kits. Halt™ Protease Inhibitor Cocktails.                             
                    Zeba™ Micro Desalt Spin Columns.
                    2-D Sample Preparation: 2-D Sample Prep for Membrane Proteins. 2-D Sample Preparation for Nuclear Proteins.
                    2-D Sample Preparation for Soluble/Insoluble Proteins. SwellGel® Blue Albumin Removal Kit. ProteoSeek™ Albumin/IgG Removal Kits.
                    2-D Protein Molecular Weight Marker Mix. SuperSignal® Chemiluminescent Substrates. Imperial™ Protein Stain. SilverSNAP® Stain II.
                    GelCode™ Blue Stain Reagent.
                    Mass Spec Sample Prep: In-Solution Tryptic Digestion and Guanidination Kit. In-Gel Tryptic Digestion Kit and PepClean™ C-18 Spin Columns.                             
                    Phosphopeptide Isolation Kit.
                    Cross-Linkers and Mass Spectrometry in Protein Structure and Protein Interaction Analysis. Deuterated Cross-Linking Reagents.
                    Cross-linking Reagents for the Analysis of Protein Interactions by Mass Spectrometry Analysis

       PIERCE:    PAGE prepTM Protein Clean-up and Enrichment Kit  (pdf)
       PIERCE:    2 D Sample Preparation for Membrane Proteins   (pdf)
       
PIERCE:    2 D Sample Preparation for Soluble or Insoluble Proteins  (pdf)
       PIERCE:    2 D Sample Preparation for Nuclear Proteins  (pdf)
       PIERCE:    SwellGel® Blue Albumin Removal Kit  (pdf) 
       PIERCE    Subcellular Protein Fractionation Kit enables stepwise separation and preparation of cytoplasmic, membrane, nuclear soluble, chromatin-bound 
                        and cytoskeletal protein extracts from mammalian cultured cells or tissue. The first reagent added to a cell pellet causes selective cell membrane  
                        permeablization, releasing soluble cytoplasmic contents. The second reagent dissolves plasma, mitochondria and ER/golgi membranes but does 
                        not solubilize nuclear membranes. After recovering the intact nuclei by centrifugation, a third reagent yields the soluble nuclear extract. A second
                        nuclear extraction with micrococcal nuclease is performed to release chromatin-bound nuclear proteins. The recovered insoluble pellet is then
                        extracted with the final reagent to isolate cytoskeletal proteins.   (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)

       QIAGEN:  Qproteome™ Bacterial Protein Preparation Handbook. For preparation of soluble proteins from bacterial cell cultures  (pdf)
       QIAGEN:  Qproteome Cell Compartment Handbook. For fast and easy subcellular fractionation of intact eukaryotic cells. By sequential addition
                         of different extraction buffers to a cell pellet, proteins in the different cellular compartments can be selectively isolated: cytosolic proteins
                         plasma membranes and compartmentalized organelles, such as nuclei, mitochondria, endoplasmic reticulum (ER), and cytoskeletal
                         proteins.   (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)  

       QIAGEN:  Qproteome Mammalian Protein Prep Kit provides gentle but efficient detergent-based lysis of mammalian cells to deliver a total protein
                         fraction suitable for any downstream application.   (pdf)

       QIAGEN:  Qproteome™ Mitochondria Isolation Handbook. For purification of mitochondria from eukaryotic cells.  (pdf)
       QIAGEN:  Qproteome™ Nuclear Protein Handbook. For isolation of nuclear and nucleic-acid binding proteins from eukaryotic cell lysates
                         (pdf-I)
 (pdf-II)
       QIAGEN   Phosphoprotein Purification Kit. For purification and analysis of phosphorylated proteins from eukaryotic cells     (pdf-I)   (pdf-II)
       QIAGEN:  Qproteome™ Plasma Membrane Protein Handbook. For fractionation of plasma membrane proteins from adherent cell culture
                `        samples. The kit use a  ligand specific for molecules on the plasma membrane vesicles that allow their elution under native conditions
                         after ligand–vesicle complexes are precipitated using magnetic beads that bind to the ligand. (pdf)

        QIAGEN:  Qproteome Soluble Protein Separation Handbook. For fractionation of protein samples used in proteomics analysis. Sequential addition
                          of increasing amounts of Fractionation Buffer precipitates proteins that can be separated by centrifugation.  (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)  
(pdf-II).  Protein Depletion Guide (pdf-III)  
       VIVASCIENCE   Removal of DNA from lysates of E. coli cells and French bean tissue prior to electrophoresis.   (pdf) 
       VIVASCIENCE   Vivapure® Anti-HSA Kit for Human Albumin Depletion 
                    Highly specific human albumin depletion with unique antibody fragments  (pdf-I)   (pdf-II)

                    Vivapure Anti-HSA Affinity Resin  (pdf-III)     

        Universal sample preparation method for proteome analysis (2009)  Jacek R Wis´niewski, Alexandre Zougman, Nagarjuna Nagaraj & 
            Matthias Mann  NATURE METHODS  VOL.6 NO.5 pg.:359  
(pdf)
           Describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass
  
           spectrometry–based proteomics
 

       Protein Precipitation Methods to concentrate or eliminate interferences before electrophoresis or protein determination

Gel Stain

     ADVANSTA    AdvanStain Scarlet is a fluorescent stain for gels and blots that allows sensitive and quantitative visualization of proteins;
            imaged on any fluorescence imaging system, including laser- and CCD-based systems, with a detection limit of less than 1 ng of protein
            per band or spot. Fully compatible with downstream Western blotting or mass spectrometry
 (pdf-I)  (pdf-Manual) 

     GE-HEALTHCARE   Deep Purple™ Total Protein Stain  (pdfI)   (pdfII)    (pdfIII)
            Deep Purple Total Protein Stain is a ultrasensitive fluorescent protein stain that can be successfully applied to both 1-D and 2-D
            electrophoresis gels. Imaging of the gels can be performed  with a variety of instrumentation. Deep Purple has significant benefits
            in comparison with other traditional protein stains such as Sypro Ruby and silver staining, including a potential increase in sensitivity
            with clear and even background. It is particularly suitable for quantitation and amenable for use with 2-D image analysis software programs.

      GE-HEALTHCARE  Fluorescent Protein Gel Stain  (pdf)  
            SYPRO™ Orange, Red, Ruby, and Tangerine protein gel stains are one-step fluorescent stains optimal for rapid and efficient staining
            of one-dimensional (1-D) protein gels. These stains provide sensitivity that is equivalent to the silver staining method for 1-D gels (1-2ng/band)
            and enable protein detection that is not affected by the presence of nucleic acids and lipopolysaccharides. SYPRO Ruby stain is also suitable
            for two-dimensional (2-D) protein gel staining  

      GE-HEALTHCARE  Silver Stain Kit (pdf)    
            A reagent kit for the fast, easy, reproducible and sensitive staining of proteins in non-denaturing gels, denaturing gels containing SDS
            and/or urea, and isoelectric focusing (IEF) gels. Allows detection of most proteins down to the nanogram range, which is 100 times
            more sensitive than Coomassie Brilliant Blue.

      GE-HEALTHCARE  SYPRO Orange and Red Protein Stain Gel  (pdf)  
            SYPRO Orange and Red protein gel stains are designed for fast, simple, sensitive staining of proteins in electrophoretic gels. Detection
            of 1-2ng of protein per minigel band; more sensitive than Coomassie™ Brilliant Blue and as sensitive as silver staining. Staining complete
            in <1 hour. Can be used with standard 300nm UV transilluminator or a laser scanner. Are not suitable for staining proteins on blotting
            membrane or in IEF gels and they show reduced sensitivity when staining proteins on 2-D gels. 

      GE-HEALTHCARE  SYPRO Tangerine Protein Stain Gel  (pdf)   
            Is designed for fast (less than 1hour), simple, sensitive staining of proteins in electrophoretic gels (4-8ng protein / band). Proteins stained
           without fixation can be used for zymography (in-gel enzyme activity) assays and can also be eluted from gels and used for further analysis
            like mass spectrometry. Allow visualization of proteins before proceeding with Western blotting.

      BIORAD:  Colloidal Gold Protein Stain   (pdf)   
            A stabilized gold solution optimized for rapid and sensitive identification of proteins bound to nitrocellulose membranes.
      BIORAD:  SYPRO Orange Protein Stain   (pdf)  
            Provides a reliable and reproducible fluorescent alternative to silver and Coomassie® staining (for SDS, native, and 2-D applications),
            with sensitive in the range of 1–10 ng per band. Requires only 30-60 minutes of staining in a single step. Will not stain DNA or RNA
            contaminants. It is also cost effective, because it is reusable.

      BIORAD:  SYPRO Ruby Protein Stains Instruction Manual (protein gel, protein blot and IEF stain)  (pdf)   
            The fluorescent stain, SYPRO Ruby protein stain is available in three application specific formulations: 1) the SYPRO Ruby protein gel
            stain for staining proteins in acrylamide gels (1-D, 2-D, isolectric focusing); 2) the SYPRO Ruby protein blot stain for staining proteins on
            nitrocellulose or PVDF membranes; 3) the SYPRO Ruby IEF stain for staining proteins in acrylamide gels separated by isoelectric focusing (IEF).
            These formulations have been optimized specifically for each application and the stains are not interchangeable.  The stain does not interfere with
            subsequent analysis of proteins by Edman-based sequencing or mass spectrometry.
       
CLONTECH:  Glycoprotein Westen detection kit   (pdf)
       EXPEDEON   InstantBlue™ is a ready-to-use, proprietary Coomassie® stain that is specially formulated for ultra-fast, sensitive and safe detection
            of your proteins. Protein gels can be stained in minutes without the need to wash, fix or destain. (pdf)  
       G-BIOSCIENCES   Protein Gel Stain Selection Guide: LabSafe GEL Blue™ - FASTsilver™  - FOCUS FASTsilver™  - RAPIDstain™  -
            Reversible Copper Stain™  - Reversible Zinc Stain™  -  Glycoprotein Staining Kit    (pdf)  
       GEBA  See Band Protein Staining Solution  (pdf) 
            Sensitivity: 38 ng protein per band.Washing uses water only.
      INVITROGENCoomasie FluorTM Orange Protein Gel Stain  (pdf)
            Fast, simple, sensitive staining of proteins in electrophoretic gels (around 8 ng of protein per band). Stained proteins can be visualized using a
            standard 300 nm UV transilluminator or a laser-based scanner. Is not recommended for staining proteins in 2-D, IEF, nondenaturing gels,
            or blotting membranes. Do not stain nucleic acid or lipopolysaccharide.

      INVITROGENOligohistidine Gel Stain  (pdf-I)  (pdf-II)  (pdf-III)   
            A fluorescent dye selective for oligohistidine fusion proteins that can be used directly in an SDS.polyacrylamide gel, eliminating the need to blot
            the protein to a membrane. Staining is complete within hours, and as little as 15 ng of a hexahistidine fusion protein can be detected. After
            documenting the oligohistidine signal, the gel can be stained for total protein using SYPRO ® Ruby protein gel stain.

      INVITROGENPro-Q Amber for selective identification of transmembrane proteins in gel   (pdf)   
            The stain preferentially stains proteins containing two or more transmembrane domains. After visualizing transmembrane proteins, the total
            protein profile can be detected using the SYPRO Ruby protein gel stain. Is not recommended for staining proteins in 2-D, IEF or
            nondenaturing gels and is not suitable for staining proteins on blotting membranes. As little as 10 ng of bacteriorhodopsin can be detected.

      INVITROGENPro-Q Diamond Phosphoprotein Gel Stain   (pdf)   
            A simple, direct method for specifically staining phosphoproteins in polyacrylamide gels. This stain can be used with standard SDS polyacrylamide
            gels or also compatible with mass spectrometry. The stain allows detection of phosphate groups attached to tyrosine, serine or threonine residues
            and can be visualized using a variety of scanning instruments. The sensitivity limit ranges from ~1–16 ng/band depending on the phosphorylation
            state of the protein.

      INVITROGENPro-Q Emerald Glycoprotein Stain Kit   (pdf)   
            Fast, simple and more sensitive than any other nonradioactive glycoprotein staining technique (it is possible to detect as little as 300 pg of
            glycoprotein per band, depending on the degree of glycosylation). Compatible with SYPRO Ruby protein gel stain for dichromatic staining.
            Stained proteins can be visualized using either UV illumination or a laser scanner.

      INVITROGENPro-Q Emerald 300 Lipopolysaccharide Stain Kit   (pdf)    
            For detection of lipopolysaccharides (LPS) in gels, with a sensitivity at least 50 times that of silver staining (as little as 2 ng of LPS per gel lane in
            just a few hours). Stains the periodate-oxidized carbohydrates of LPS with a bright green fluorescence that is easy to visualize using a simple UV  
           transilluminator. Contaminating proteins can be easily visualized on the same gel by using the orange-red–fluorescent SYPRO Ruby protein gel stain.

      INVITROGENSYPRO® Orange and SYPRO® Red Protein Gel Stains (pdf) 
            Can detect 4–8 ng of protein. Not suitable for staining proteins on blotting membranes and reduced sensitivity when staining proteins in IEF or 2-D gels.
      INVITROGENSYPRO Rubin Protein Gel Stain   (pdf) 
            Created especially for the analysis of proteins in 2-D polyacrylamide gels. It provides the same low nanogram sensitivity as the best silver staining
            techniques, but stains more proteins and has a much broader linear quantitation range. Staining is compatible with mass spectrometry or
            microsequencing. Can be visualized with UV transilluminators or laser scanners.

      PERKIN ELMER  Phos-tag™ Gel and Blot Stains for Detection of Protein Phosphorylation Phos-tag™ is a novel chelate with unparalleled selectivity
            and sensitivity for phosphoproteins. It exhibits excellent affinity for phosphomonoesters of tyrosine, serine and threonine. Phos-tag™ can be linked
            to a variety of fluorophores, allowing a range of choices in spectral properties and detection hardware. Sensitivity to ˜ 1 ng phosphoprotein

            (pdf-I)   (pdf-II)
      PIERCE:   Gel Code Blue Stain Reagent   (pdf)   
            Utilizes the colloidal properties of coomassie G-250 dye for protein staining on polyacrylamide gels. Stains only protein and allows bands to be
            viewed directly on the gel during the staining process. After staining, a water equilibration step (Water Wash Enhancement™) further enhances
            staining sensitivity and yields a clear background.

      PIERCE:   Gel Code Color Silver Stain Kit  (pdf)  
            An improved formulation of the Silver Stain procedure. Has been successfully used to stain proteins, DNA, RNA, lipids and polysaccharides.
            Along with the quantitation of protein it also characterizes protein via color. This color characterization distinguishes overlapping proteins, allows
            for increased pattern recognition and visualization, and identifies minor contaminants in samples.

      PIERCE:   Gel Code E-ZincTM Reversible Stain Kit   (pdf)
      PIERCE:   Gel Code Glycoprotein Staining kit  (pdf)
      PIERCE:   Gel Code Phosphoprotein Staining Kit   (pdf)  
      PIERCE:   Gel Code Silver SNAPTMStain Kit  (pdf)
      PIERCE:   GelCode Stains Technical Review   (pdf)
      PIERCE:   Imperial™ Protein Stain is a coomassie R-250 dye-based reagent for protein staining in polyacrylamide gels. This reagent stains only
            protein and allows bands to be viewed directly in the gel during the staining process (≤ 3 ng). The easy protocol is flexible to meet demanding
            time and sensitivity requirements and uses a simple water wash to yield a clear background.  (pdf)

      PIERCE:   Insta StainTM Blue Gel Stain paper  (pdf)    
      PIERCE:   Mem CodeTM Reversible Protein Stain Kit  (pdf)   
      PIERCE:   Krypton™ Glycoprotein Staining Kit enables fast and sensitive fluorescent staining for the detection of glycoproteins. The stain is sensitive
            down to 15 ng of glycoprotein per band, depending upon the nature and degree of glycosylation on the proteins.  (pdf)

      PIERCE:   Krypton™ Infrared Protein Stain enables sensitive fluorescent visualization of proteins separated by 1-D or 2-D SDS-PAGE.
            Sensitivity 0.25 ng using the standard protocol or down to 1 ng using the rapid 60 minute protocol. The stain has an excellent dynamic range and
            is compatible with subsequent analysis by mass spectrometry.  (pdf)

      PIERCE:   Silver Stain Rescue Reagent. Silver Stain Rescue Reagent enables removal of undesirable background from unevenly and overly
            developed silver-stained gels.  (pdf)

      SIGMA    Protein Staining Reagents for electrophoresis - Protein Dyes and Stains Selection Chart  (pdf)   
            Colorimetric and Fluorescent Methods for detection of  proteins, glycoproteins, lipids and/or lipoproteins, nucleic acids, phospholipids,
            etc, for all kind of electrophoresis systems.

      SIGMA  Glycoproteomics Selection Guide — Labeling and Detection, Quantitation  (pdf)

      Silver Stain

Proteases

      CALBIOCHEM  Proteases Product Listing  (pdf)
      JENA BIOSCIENCE   Protease Detection Kit detects a wide variety of proteases, including serine proteases, cysteine proteases and acid proteases
          in biological samples. The kit is based on a labeled casein derivative as protease substrate  (pdf)
      MOLECULAR PROBES  Protease Assay Kit  (pdf)

      NOVAGEN  Protease Assay Kit  (pdf)
      ROCHE   Universal Protease Substrate (pdf)
     
CARDIFF UNIVERSITY - MOLECULAR CELL BIOLOGY - EHRMAN LAB   Proteases of E.Coli

Analytical Ultracentrifugation
       EMBO: Protein Expression and Purification Facility of the European Molecular Biology Laboratory  Sedimentation Equilibrium Experiment
       Alliance Protein Laboratories:  Sedimentation equilibrium is an analytical ultracentrifugation (AUC) method  for measuring protein molecular masses in 
            solution and for studying protein-protein interactions. 
It is particularly valuable for:  
                   1. establishing whether the native state of a protein is a monomer, dimer, trimer, etc.

                   2. measuring the equilibrium constant (
Kd) for association of proteins which reversibly self-associate to form oligomers
                   3. measuring the stoichiometry of complexes between two or more different proteins or between a protein and a non-protein ligand 

                   4. measuring the equilibrium constants for reversible protein-protein and protein-ligand interactions

                (Website A.P.L.)  (file)
       Alliance Protein Laboratories:
   Sedimentation velocity is an analytical ultracentrifugation (AUC) method that measures the rate at which molecules move 
            in response to centrifugal force generated in a centrifuge. This sedimentation rate provides information about both the molecular mass and the shape 
            of molecules. In some cases this technique can also measure diffusion coefficients and molecular mass. 
Sedimentation velocity is particularly valuable for:
                    1. verifying whether a sample is entirely homogeneous in mass and conformation
                    2. detecting aggregates in protein samples and quantifying the amount of aggregate
                   
                    3.
comparing the conformations for samples                     
                    4. establishing whether the native state of a protein or peptide is a monomer, dimer, trimer, etc.
                    5. determining the overall shape of non-glycosylated protein and peptide molecules in solution  
                    6. measuring the distribution of sizes in samples which contain a very broad range of sizes  
                    7. detecting changes in protein conformation, for example partial unfolding or transitions to "molten globule" states  
                    8. studying the formation and stoichiometry of tight complexes between proteins 
             
(Website A.P.L.)  (file)

Protein-Protein Interaction

     G-BIOSCIENCES   Protein Cross-Linkers Handbook & Selection Guide  (pdf)  
     IBA GmbH:   With four different determination systems based on Strep-tag•II and One- STrEP-tag, they provide optimal solutions for in vivo protein-protein 
            interaction analysis. (pdf)
     PIERCE:   Protein Interactions - Manual  (pdf)

     Mellacheruvu D. et al. (2013) Nature Methods 8: 730-736  The CRAPome: a contaminant repository for affinity purification–mass spectrometry                             data  (pdf)  CRAPome

Circular dichroism
  
Alliance Protein Laboratories:  Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed
            polarized light which arise due to structural asymmetry. The absence of regular structure results in zero CD intensity, while an ordered structure results in a
            spectrum which can contain both positive and negative signals. (Website A.P.L.)  (file)

Light Scattering

       Alliance Protein Laboratories:     (Website A.P.L.)  (file)
       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)




Entries since September 2006   


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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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