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Small scale His-Tag purification under nature conditions The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology
The Hebrew University of Jerusalem
Dr. Mario Lebendiker  Tel: 972-2-6586920 

Test Tube for Ion Exchange Chromatography
(A similar protocol could be used for Hydrophobic Exchange or Affinity Chromatography)


The purpose of the test tube is to find proper binding conditions such as pH and resin type, of the target protein (or contaminating proteins) to IEX (ionic exchange) resins.  Once you know the best pH binding conditions, a second test tube at different salt concentrations will provide information about washing and elution conditions.

1. Sample preparation
The idea is to adjust the sample to binding conditionsin 20mM buffers at different pH, and low salt concentration.
(If necessary use additives such as: EDTA; glycerol; DTT, DTE or ßME; protease inhibitors; detergents, etc.)

    Cation exchange buffers
pH range
3.8 - 4.3
Na Formate
3.5 - 5.5
Na Acetate
3.5 -6.5
Na Citrate
5.5 - 6.7
5.5 - 8.0
7.6 - 8.2
8.2 - 8.7

    Anion exchange buffers
pH range
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 adjustment of the sample can be done in different ways:
a) Dialyze samples overnight vs. 40ml of 20mM buffers
b) Dilute sample 1:10 with 20mM buffers
c) Desalting columns using 20mM buffers

2.Column equilibration
Use a) strong anion exchange: like Q-Sepharose FF and
       b) strong cation exchange: like SP-Sepharose FF.
Add 80ul resin slurry to Eppendorf tubes: i.e. several tubes with SP-Sepharose and several tubes with Q-Sepharose.
Wash each tube with water: add 1.2ml to each tube; mix; spin 3min 3500rpm; discharge supernatant.
a) Wash each Q-Sepharose tube twice with different buffers for anion exchange, using 0.5 pH unit difference between them (pH 6.5 - 9.0)
b) Wash each SP-Sepharose tube twice with different buffers for cation exchange, using 0.5 pH unit difference between them (pH 4.0- 7.5)

Add 200-300ul sample to each tube. Mix at least 30min @ 4°C. Spin and check the supernatant. Standard: A sample taken before binding to any resin.
If the protein is enough concentrated the supernatant could be checked by PAGE-SDS and Coomasie Blue Staining. If the protein is concentrated enough the supernatant could be checked by SDS-PAGE and Coomassie Blue Staining.  If necessary, concentrate the protein by acetone or TCA precipitation, or use Silver Staining.

Other possibilities: Western blot; biological assays (in this case is very important to check controls at different pH); use of radioactive labeled protein; etc.

Expect better binding at lower pH using cation exchange, and at higher pH with anion exchange.
Once you decide on the best pH binding conditions, you can check "elution conditions" by repeating the same test tube with the best pH conditions using now 20mM buffer + increasing concentrations of NaCl  (50, 100, 200, 300, 500, 750 and 1000mM) for the resin equilibration and the sample binding conditions. This second test tube will give you the best conditions for binding, washing and elution.

Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem  Tel: 972-2-6586920  

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