Test Tube for HIC: Hydrophobic Exchange Resins
INTRODUCTION
The purpose of the test tube is to found
proper binding and elution conditions (salt concentration, salt type and resin
type) of the protein target or contaminants proteins to HIC resins before
scale-up to FPLC chromatographic level. Use of HIC resins could be ideal to
perform separations according to hydrophobicity 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 and high salt concentration: 1-2 M ammonium sulphate
or 3M NaCl (or other salts).
If necessary use additives: EDTA;
glycerol; DTT, DTE or βME; protease inhibitors; etc
Take care that the protein does not
precipitate at the high salt concentration
2.Column equilibration
Phenyl,
Octyl or Butyl Sepharose form 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 200-300µl 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 decrease of [NaCl]
or [ammonium sulphate]. At least four different concentrations till buffer
alone: 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
Precipitate protein samples by Acetone or
TCA precipitation (eliminate high salt), and checked by PAGE-SDS and Coomasie
Blue Staining.
Other possibilities: Western blot;
biological assays (in this case is very important to check controls at
different [salt]); use of radioactive labeled protein; etc.
Fine
tuning optimization according to results
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