Test Tube for Ion Exchange Chromatography
(A similar protocol could be used for Hydrophobic Exchange or
Affinity Chromatography)
INTRODUCTION
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
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Anion exchange buffers
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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)
3.Binding
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.
FINAL CONCLUSIONS
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.
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Copyright
©, 2002, The Hebrew University of Jerusalem. All Rights Reserved.