Differentiation of proteins in polyacrylamide gels by a modification of silver staining for the PhastSystem and a laser densitometer.
Nonspecific background staining of the gel matrix is the limiting factor in the differentiation of proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is caused by nonspecific binding of silver ions in the gel matrix, either to nonprotein compounds or through the chemistry of the polyacrylamide gel itself. The resulting stain of the gel produces a stain baseline, making differentiation of protein bands with a laser densitometer difficult. We have therefore developed a modified silver staining method for the PhastSystem to reduce such nonspecific background staining. Apart from slowing the development program by lowering the temperature to 15 degrees C and shortening the incubation time to 4 min, the essential step in the modification is the combined use of an EDTA and Tris-acetate buffer solution which stops the reduction of silver ions. The reduced background staining leads to an improved detection of protein bands and virtually identical zero lines for the laser densitograms and the stain baselines. The total staining time is 91 min. All steps in the program are fully automated and continuous, employing the PhastSystem staining unit.
Kierdorf
H
H
Department of Internal Medicine II, Aachen University of Technology, Germany.
Melzer
H
H
Mann
H
H
Sieberth
H G
HG
D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007834:Lasers; D011506:Proteins / Q000302:isolation & purification*; D011507:Proteinuria / Q000652:urine; D016622:Silver Staining / Q000379:methods*
D011506:Proteins