INTRODUCTION

The purpose of this simple test tube is to found proper binding conditions (pH and type of resin) of the protein target or contaminants proteins to IEX (ionic exchange resins) before scale-up to FPLC chromatographic level.

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 buffers (CEIX)

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

    Anion exchange buffers (AEIX)

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

The buffer adjusting could be done by different ways:

• Dialyze samples over-night vs. 100-200 volumes of 20mM buffers
• Dilute sample 1:10 with 20-50mM buffers
• Desalting columns using 20mM buffers

Start conditions for AEIX resins: use pH buffers 2 -3 units higher than the protein pI and 0-100mM NaCl. Increase starting pH in case you need higher [NaCl] to maintain protein stability

Start conditions for CEIX resins: use pH buffers 2 -3 units lower than the protein pI and 0-100mM NaCl. Decrease starting pH in case you need higher [NaCl] to maintain protein stability

2.Column equilibration
Strong anion exchange: like Q-Sepharose FF from GE or other companies
Strong cation exchange: like SP-Sepharose FF from GE or other companies
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 resin tube. Mix gentle 30min 4°C.  Spin 3min 3500rpm. Keep supernatant: unbound.
B) Wash with 200ul start condition buffer: wash.
C) Elution: successive increase of [NaCl], like 100, 250, 500, 750 and 1M NaCl in buffer: elution
D) Add 200µl of PAGE-SDS sample buffer to resin, heat two minutes at high temperature, mix, spin and keep supernatant: resin

 
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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