Protocols and tips in protein purification or How to purify protein in one day. Some protein purification protocols include a treatment of the crude extract. This could be dialysis, differential ultra centrifugation, addition of a reagent (such as substrates or inhibitors, etc.) or precipitations of some components of crude extract using different precipitants.
Dialysis is normally required if the first chromatographic step should be performed at a different pH.
Differential ultra centrifugation is applicable if the target protein (TP) is associated with a certain part of the cell or with certain organelles. It is not necessary for purification of soluble cytoplasmic proteins.
Fractionation of the crude extract by various precipitants was a very popular and rather powerful method in protein purification in the early days when chromatographic techniques were not properly developed. Ether, chloroform, ethanol, isopropanol and poliols were widely used. Nowadays ammonium sulphate is practically the only precipitant that is frequently used for fractionation of the crude extract (in a so called ammonium sulphate cut procedure).
Ammonium sulphate cut (see Protocols section for the procedure)
For this application ammonium sulphate concentration is usually expressed in a % of saturation. Saturated solution (100%) at room temperature is about 4.1M. Using the Ammonium sulphate table (see Tables and Charts section) you can find out how much ammonium sulphate powder you should add to the solution to get a desirable concentration.
During precipitation the protein solution should be kept on ice or at 4?C to prevent proteins from denaturing due to heat produced by the ammonium sulphate dissolving. After this step the protein sample will contain significant amounts of ammonium sulphate and can not be used directly for ion exchange chromatography but can be used for hydrophobic chromatography (after adjusting ammonium sulphate concentration to required level) or applied on the gel filtration column for separation or desalting.
Clarification of crude extract by protamin sulphate treatment is another method which can sometimes be useful. Protamin sulphate is used to precipitate out nucleoproteins such as ribosomes and DNA/protein complexes. In my work it has been particularly useful for the clarification of fungal extract. After PS clarification the sample can be used directly for any kind of chromatography (see Protocols sections for the procedure).
Denaturing of contaminating proteins by heating has become a rather common procedure which is especially effective if you are dealing with recombinant protein from a thermophilic organism expressed in E.coli . If you know the denaturing temperature of the TP, you can heat to 5-10 degrees lower then that for 10-20 minutes. This process depends on pH, salt concentration and protein concentration. Small scale trials are recommended to optimise all conditions.