Preparation of the chromatographic columns

 - Protein crystallography

Main steps:

   - Protein purification
     - Introduction
     - Step-by-Step
       - Equipment & Reagents
       - Stock solutions
       - Chromatographic columns
       - Crude extract
       - pre Chromatographic step
       - Chromatographic step
     - Common sense
     - Protocols
     - Charts & Tables
     - Appendix
   - Crystallisation


   - Programs for crystallography
   - X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

   - CCP4BB

Protocols and tips in protein purification or How to purify protein in one day.

Preparation of the chromatographic columns

For the basic purification protocol you need a set of 3 columns packed with
DEAE-Sepharose Fast Flow (weak anion exchanger)
Phenyl-Toyopearl 630S (hydrophobic matrix)
Superdex-200 (gel filtration)

The additional set includes:

Q-Sepharose Fast Flow (strong anion exchanger)
CM-Sepharose fast flow (weak cation exchanger)
DEAE-Toyopearl 650S ( weak anion exchanger)
SP-Toyopearl 650S (strong cation exchanger)
Butyl-Toyopearl 650S (strong hydrophobic matrix)
Ethyl-Toyopearl 650S (weak hydrophobic matrix)
Heparin-Sepharose Fast Flow (pseudo-affinity)
You may need other types of matrices, such as Hydroxylapatite, Dye matrix, etc.

You can buy ready made columns from Amersham or Bio-Rad. For our scale we need columns 10-30 ml in size. I prefer to make them myself as this is cheaper and makes them repackable.

I like Amersham-Pharmacia empty columns C-type and XK-type with adapters. The sizes are 1x20 cm and 1.6x20 cm.

To prepare any column (except the gel filtration one):
  1. Check that all parts of column are in place (according to specification)
  2. Fix empty column on a stand in vertical position
  3. Fill with few ml of ultra pure water
  4. Prepare slurry of any matrix taking 1 part sediment matrix and two parts water, warm to room temperature if matrix has been kept in the fridge. Degasation under vacuum is useful but not overly crucial.
  5. Having the bottom end of column open, pour the slurry into the column, adding the slurry portion by portion, the matrix should settle at the bottom of the column and the water should run through. When the matrix has settled - close the bottom of the column.
  6. Completely fill column with water. Fill an adapter with water and fit it onto the top of the matrix bed. Press down slightly and close the adapter.
For gel filtration it is the best to use a pre-packed column, this is because a self made column never gives as good a separation as a ready made one.

A 1.6x60 cm Hi-Load Superdex 200 column (Amersham-Pharmacia) is the best option for the majority of proteins.