INTRODUCTION

The purpose of the test tube is to found proper binding and elution conditions (pH and type of resin) of the protein target or contaminants proteins to MM (mixed mode resins).
This simple test tube is ideal to find proper MM conditions at low scale before scale-up to FPLC chromatographic level. Use of MM resins could be ideal to perform charge separations to proteins that are non-stable at low salt concentrations (some prone to aggregate proteins).

1. Sample preparation
The idea is to adjust the sample to binding conditions: 20mM buffers at different pH, and low salt concentration.
(If necessary use additives: EDTA; glycerol or other non-ionic osmolytes or additives; DTT, DTE or βME; protease inhibitors; non-ionic or zwiterionic detergents, etc.)

Cation exchange MM buffers: Capto™ MMC: a multimodal cation exchanger (GE) or TOYOPEARL MX-Trp-650M from TOSOH
Use minimal salt concentration that allows your protein to be in solution

pH range	Buffer
3.8 - 4.3	Na Formate
3.5 - 5.5	Na Acetate
3.5 -6.5	Na Citrate
5.5 - 6.7	MES
5.5 - 8.0	Phosphate
7.6 - 8.2	HEPES-HCl
8.2 - 8.7	Bicine-HCl

For Anion exchange MM bufers: Capto adhere from GE or Pall HEA and PPA HyperCel 
Use minimal salt concentration that allows your protein to be in solution

pH range	Buffer
4.5 - 5.0	N-methylpiperadine HCl
5.5 - 6.0	Histidine HCl
5.8 - 6.4	Bis-Tris HCl
6.5 - 7.7	Imidazole HCl
7.3 - 9.0	Triethanolamine HCl
7.5 - 9.0	Tris HCl

Start conditions for Anion exchange MM resins: use pH buffers 2 -3 units higher than the protein pI and ~200-300mM NaCl. Increase starting pH in case you need higher [NaCl]
Start conditions for Cation exchange MM resins: use pH buffers 2 -3 units lower than the protein pI and ~200-300mM NaCl. Decrease starting pH in case you need higher [NaCl]

Buffer adjusting could be done by different ways:

• Dialyze samples over-night vs. 100-200 volumes of 20mM buffers + NaCl
• Adjust sample pH adding x 5 concentrated buffer pH and adjust NaCl concentration by dilution (if [NaCl] is high) or adding concentrated NaCl (if [NaCl] is low)
• Desalting columns using 20mM buffers + NaCl

2.Column equilibration
Add 30ul slurry to Eppendorf tubes
Wash each tube with water: add 1.2ml to each tube; mix; spin 3min 3500rpm; discharge supernatant.
Wash twice with start condition buffer.

3.Experiment
A) Add 200-300ul sample to each tube. Mix gentle 30min 4°C.  Spin 3min 3500rpm. Keep supernatant: unbound.
B) Wash with 200ul start condition buffer: wash.
C1) Elution for Anion exchange MM resins: successive decrease of one unit pH with or without increasing [NaCl]. At least four different pH units: elution
C2) Elution for Cation exchange MM resins: successive increase of one unit pH with or without increasing [NaCl]. At least four different pH units: elution

 
4. How to check
If the protein is enough concentrated the supernatant could be checked by PAGE-SDS and Coomasie Blue Staining. If necessary, concentrate protein by Acetone or TCA precipitation, or use Silver Stain.
Other possibilities: Western blot; biological assays (in this case is very important to check controls at different pH); use of radioactive labeled protein; etc.

Fine tuning optimization according to results

 

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