Protein Extraction

The first step is to isolate the proteins from the system of interest. Proteins need to be separated from salts, nucleic acids, lipids, polysaccharides, phenolics, and other cellular material. The main purpose of this step is to eliminate or minimize the interfering materials in the downstream workflow and it could be the most challenging step. If targeted proteomics approach is desired, the isolation of cellular target is done before protein extraction.

Extraction procedure usually involves cell disruption to release the proteins from the cell combined or followed with precipitation procedures. If sample source is liquid (blood cells, tissue culture cells) gentle lysis can be performed. For solid samples vigorous lysis should be used. There are many sample preparation methods available. The choice of protein extraction method largely depends on the expected outcome of the research, yet it is crucial to the success of experiments.

TCA Acetone precipitation
(Tsugita A, Kawakami T, Uchiyama Y, Kamo M, Miyatake N, Nozu Y, Electrophoresis 1994, 15: 708–720)

Methanol chloroform precipitation
(Wessel D, Flügge UI, Analytical Biochemistry 1984, 138: 141-143)

Tris buffer extraction
(Molloy MP, Herbert BR, Walsh BJ, Tyler MI, Traini M, Sanchez J-C, Hochstrasser DF, Williams KL, Gooley AA, Electrophoresis 1998, 19: 837–844)

Sequential extraction
(Lehner I, Niehof M, Borlak J, Electrophoresis 2003, 24: 1795-1808)

After this point the protein mixture could be separated with HPLC methods such as such as reverse phase, size exclusion chromatography if 2D gel electrophoresis is not desired.

For additional information, contact Victor Asirvatham.