Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis.
A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.
Sørensen
Birgitte Kjaer
BK
Department of Research and Development, Statens Serum Institut, Artillerivej 5, DK-2300, Copenhagen S, Denmark.
Højrup
Peter
P
Østergård
Erik
E
Jørgensen
Charlotte Svaerke
CS
Enghild
Jan
J
Ryder
Lisa Rebekka
LR
Houen
Gunnar
G
D002460:Cell Line; D003101:Collodion; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004727:Endothelium / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins / Q000302:isolation & purification*; D012834:Silver; D013194:Staining and Labeling / Q000379:methods*; D005465:Fluorocarbon Polymers
D003101:Collodion; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D012834:Silver; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers