Quantitation of electrophoretic eluted proteins.
Quantitation of stained, electroeluted proteins by the classical Lowry and Bradford protein assay is not possible because of some different interferences. In particular we have found that the substance interfering in the Lowry method cannot be removed by trichloroacetic acid precipitation nor can be compensated for by the appropriate blank. Interferences in the Bradford protein assay are due to detergents and pH of the protein buffer as well as to Coomassie brilliant blue R250 electroeluted with the protein sample. However, while these interferences can be compensated for by appropriate blank and standard curves, others (probably due to acrylamide fines) cannot be corrected. All these problems can be overcome by concentration and dialysis of electroeluted samples which permit the removal of interfering substances and the use of Bradford and Lowry protein assay in the 1-20 micrograms range, respectively. Successful applications are described for electroeluted bovine serum albumin, human hexokinase and phosphoglucomutase.
Magnani
M
M
Istituto di Chimica Biologica, Università di Urbino.
Stocchi
V
V
Serafini
G
G
Chiarantini
L
L
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D012394:Rosaniline Dyes; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate
D011506:Proteins; D012394:Rosaniline Dyes; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; C048139:Coomassie blue