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Recovering of protein from the ammonium sulphate precipitate

- Protein crystallography

Main steps:

   - Protein purification
     - Introduction
     - Step-by-Step
     - Common sense
     - Protocols
       - Stock solutions
       - Cell disruption
       - Protamin sulphate treatment
       - Analytical AM cut
       - Preparative AM cut
       - Protein precipitation by AM
       - Protein recovery from AM
       - Solubility of expression
       - Solubility of expression
       - Protein concentration
       - Protein concentration
     - Charts & Tables
     - Appendix
   - Crystallisation

Special:

   - 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.

  1. Place an appropriate volume of the ammonium sulphate precipitate suspension into a centrifugation tube.
  2. Collect precipitate by centrifugation at 5000 g or higher for 10 min at 4°C.
  3. Remove supernatant fraction carefully, try to take out every last drop of ammonium sulphate solution.
  4. Re-suspend pellet in a small volume of appropriate buffer and dialyse it against some 100-200 volumes of buffer. Make 2-3 changes, the last one should be left overnight for full equilibration. After 2 changes you can expect there to be about 1-2mM of ammonium sulphate left in the sample, with 3 changes it is close to 0.
  5. It is useful to clarify sample from possible insoluble contaminants by centrifugation at 25000g or higher for 15 min.
NB: Remember that some proteins can not be recovered from ammonium sulphate precipitate.

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