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Consecutive silver staining and autoradiography of 35 S and 32 P‐labeled cellular proteins: Application for the analysis of signal transducing pathways
Author(s) -
Luo Lindi,
Wirth Peter J.
Publication year - 1993
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150140121
Subject(s) - staining , silver stain , dephosphorylation , phosphorylation , methionine , polyacrylamide gel electrophoresis , gel electrophoresis , microbiology and biotechnology , coomassie brilliant blue , differential staining , biochemistry , biology , chemistry , amino acid , phosphatase , enzyme , genetics
The methodology for the simultaneous analysis of protein synthesis concomitant with protein phosphorylation/dephosphorylation is described. The technique consists of metabolic labeling of rat liver epithelial (RLE) cells with [ 32 P]orthophosphate and [ 35 S]methionine, performing two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) of the mixed samples, followed by silver staining and subsequent autoradiography of the dried silver stained 2‐D PAGE electrophoretograms using two films placed back‐to‐back. The first film, which is positioned in direct contact with the dried silver‐stained gel, visualized both exposure to 35 S and 32 P while the second film recorded exposure to only 32 P due to the differential energy levels of the two isotopes. The juxtapositioning of the silver‐stained images with the two autoradiographic film images permits the unambiguous mapping of the phosphorylated polypeptides back to their corresponding silver‐stained and methionine‐labeled counterparts. This strategy provides quantitative information utilizing both silver staining (measure of constitutive levels of protein expression) and metabolic labeling to measure rates of protein synthesis and/or degradation and phosphorylation and/or dephosphorylation using [ 35 S]methionine and [ 32 P]orthophosphate, respectively. We have utilized this methodology for the in vitro analysis of transforming growth factor type β 1 (TGF‐β 1 )‐mediated signal transduction in RLE cells and have identified three nuclear polypeptides, 1 (p I 4.95/ M r 97 kDa), 2 (5.00/85 kDa) and 3 (4.90/84 kDa) whose phosphorylation status is rapidly and transiently modulated by TGF‐β 1 . The methodology described should have wide applications in studies where it is desirous to measure protein synthesis and/or degradation concomitant with signal transduction pathways involving protein phosphorylation.