Consecutive silver staining and autoradiography of 35S and 32P-labeled cellular proteins: application for the analysis of signal transducing pathways.
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 [32P]orthophosphate and [35S]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 35S and 32P while the second film recorded exposure to only 32P due to the differential energy levels of the two isotopes. The juxta-positioning 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 [35S]methionine and [32P]orthophosphate, respectively. We have utilized this methodology for the in vitro analysis of transforming growth factor type beta 1 (TGF-beta 1)-mediated signal transduction in RLE cells and have identified three nuclear polypeptides, 1 (pI 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-beta 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.
Luo
L D
LD
Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Wirth
P J
PJ
D000818:Animals; D001345:Autoradiography; D003201:Computers; D005260:Female; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D051381:Rats; D011916:Rats, Inbred F344; D015398:Signal Transduction; D016622:Silver Staining; D013462:Sulfur Radioisotopes; D016212:Transforming Growth Factor beta / Q000502:physiology
D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D016212:Transforming Growth Factor beta