Silver staining of gels

Fig. 2 (a) Silver stain of gel electrophoresis of pure recombinant rat PAP (size in kDa). Lane 1 native PAGE. Lane 2. SDS-PAGE. (b) Immunodetection (size in kDa) of purified recombinant rat PAP by Western-blotting. Lane / native PAGE. Lane 2. SDS-PAGE. Immunodetection with rabbit polyclonal antibody against an N-terminal peptide of rat PAP (in house antibody) [3]... 

The purity of the AE fraction was investigated by SDS-PAGE using Pharmacia PhastSystem with 10 - 15% SDS-gradient gels. Electrophoresis and silver staining of the proteins were performed as described by the manuals from Pharmacia. For determination of pi lEF 3-9 PhastSystem gels were used. 

Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850—858. 

In silver staining, the gel is Impregnated with soluble silver ions and developed by treatment with formaldehyde, which reduces silver ions to form an insoluble brown precipitate of metallic silver. This reduction is promoted by protein. 

After silver staining, the gel is thoroughly washed by several changes of ddH20 (at least four changes of ten gel volumes, 10 min... 

Support Protocol 2 Silver Staining of Polyacrylamide Gels B3.1.15... 

Heukeshoven, J. and Dernick, R. 1985. Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6 103-112. 

Fig. 7. Evidence for protein phosphorylation as an early event in the psi response. 2 ml of three-day-old cells growing —Pi or +Pi were washed for 2 h in +Pi consisting of 20 pCi of carrier-free (32P)-orthophosphate. Cells were pelleted and total proteins extracted and separated via SDS-PAGE. A, Autoradiogram with equal cpm loaded per lane. The arrow indicates the position of a putative phosphorylated protein that showed psi enhancement. B, The same as A except that the lanes were loaded to represent equal amounts of cell biomass (dry weight). Dramatic enhancement in phosphorylation of all bands in B may be the result of increased rates of Pi transport in the —Pi treatment or these data may imply that several proteins show enhanced phosphorylation under psi conditions. C, As A and B except total proteins were visualised via silver staining. (All gels are aligned by apparent molecular mass.)...

Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., and Imai, B. S. (1999) Mass spectrometric identification of proteins from silver-stained polyacrylamide gel a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20, 601-605. 

A, Shevchenko, M. Wilm, O. Vorm, M. Mann, MS sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem., 68 (1996) 850. 

---