Test Tube for HIC: Hydrophobic Exchange Resins
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
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.
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.
tuning optimization according to results
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