Preparation of crude extract

 - 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.

Disruption of bacterial cells

Methods for cell disruption can be divided into two groups:

  1. Non mechanical methods, including; freezing-thawing, osmotic shock, lysozyme treatment
  2. Mechanical methods, including ultrasonication, liquid extrusion (French Press)

For full release of the soluble cytoplasmic proteins a combination of non-mechanical and mechanical methods should be employed. For example it is widely recommended to apply lysozyme treatment followed by ultrasonication. I've found a combination of using osmotic shock, freezing-thawing and sonication simpler and quicker.

For effective sonication, cell suspensia has to be prepared by adding 8-20 ml of buffer per gram (or ml) of cell pellets. During sonication cell suspensia should be placed in an ice bath.

To prevent the protein denaturing due to the heat released by the probe, sonication should be performed in 3/4 cycles for 20-25 sec at 16-micron amplitude (the maximum on a Soniprep 150 machine), allowing the sample to cool between sonication sessions.

Tip: To save time on this step it is useful to divide cell suspension into 3-4 portions and treat the portions one after another thereby allowing each portion to cool while you treat the other portions. Also, you can add small peaces of ice in to cell suspension between the sonication sessions.

Removal of cell debris (insoluble part of cells)

Practically the only method used for the removal of debris is centrifugation.

A method called differential centrifugation can be applied to separate a homogenates components by size.
  • To spin down unbroken cells and other large bits apply 10 000g for 5-10 minutes.
  • To pellet inclusive bodies apply 10 000g for 10 min.
  • To collect membrane fractions 1 hour at 100 000g is required.
  • To pellet ribosomes apply 100 000g for 3 hours.

Normally to purify soluble cytoplasmic proteins we do not need to remove ribosomes or all the membranes from the crude extract, we only need to remove big particles (bigger than 10µm, which is the mesh size of the net used in a chromatographic column adaptor).

Empirically the optimal protocol has been found to be as follows:
  • For JA-20 rotor at J-20 centrifuge, set 19000 rpm (about 43000g) for 15 minutes
  • For JA-25.5 rotor (red) at J-25 Avanti centrifuge, set 24000 rpm (about 70000g) for 10 min at 4°C

For purification of membrane proteins you only need to spin down the large pieces (this is done by centrifugation at 10000g for 10 minutes) and then membranes need to be collected, this is done by ultracentrifuge at 100000g for 1 hour.