A new two-step precipitation method removes free-SDS and thiol reagents from diluted solutions, and then allows recovery and quantitation of proteins.
Several polypeptides are present in a few copies per cell (i.e., membrane receptors or transcription factors), and therefore their concentration and quantitation from highly diluted solution after detergent solubilization is often an essential and difficult step in their purification by gel electrophoresis. A solution (optimized to Tris-glycine, Tris-borate, and Na-phosphate buffers) to all of these problems is here described, detailing a two-step procedure which takes advantage of the lower solubility of free potassium dodecyl sulfate (KDS) vs micellar KDS even at neutral pH. In the first step, more than 90% of free DS precipitates out from protein solution, the method being poorly sensitive to dodecyl sulfate/KCl ratio from almost no SDS to 10%. Indeed to obtain a residual 0.01% of SDS in the solution no adjustment of KCl concentration is needed from 0.1 to 2.3% SDS. When the supernatant is added with ice-cold TCA and K+, protein solubility is severely affected. Excellent protein recovery, at least 90%, is obtained with hydrophilic proteins. On the other hand, hydrophobic or low-ionic-strength-insoluble proteins are partially lost in step 1 precipitate, so that protein yield decreases, still remaining to about 80%. Furthermore our procedure simplifies determination of protein contents of diluted mercaptoethanol-SDS-solubilized proteins: since thiol reagents are discarded with the final supernatant, proteins can be quantitated in the final pellet by standard colorimetric methods.
Carraro
U
U
Department of Experimental Biomedical Sciences, University of Padua, Italy.
Doria
D
D
Rizzi
C
C
Sandri
M
M
D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D008099:Liver / Q000737:chemistry; D009132:Muscles / Q000737:chemistry; D009218:Myosins / Q000032:analysis / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate / Q000302:isolation & purification; D012995:Solubility; D012996:Solutions; D013439:Sulfhydryl Reagents / Q000302:isolation & purification
D011506:Proteins; D012996:Solutions; D013439:Sulfhydryl Reagents; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; C028913:dodecyl sulfate; D009218:Myosins