Consecutive silver staining and autoradiography of 35S and 32P-labeled cellular proteins: application for the analysis of signal transducing pathways

Electrophoresis
L D Luo, P J Wirth

Abstract

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 dephosphory...Continue Reading

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Citations

Nov 1, 1993·Electrophoresis·P J WirthD C Parmelee
Jun 21, 2011·Journal of Chemical Biology·Victoria J GauciJens R Coorssen
Jul 4, 2006·NeuroRx : the Journal of the American Society for Experimental NeuroTherapeutics·Leila H ChoeKelvin H Lee
May 23, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Wayne F Patton
Nov 23, 2006·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Anna Bodzon-KulakowskaJerzy Silberring
Jul 31, 2013·Proteomics. Clinical Applications·A B SivaSisinthy Shivaji
Oct 14, 2005·Bioscience Reports·Roberto RaggiaschiGeorg C Terstappen
Jan 1, 1996·Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology·D Kültz, G N Somero
Apr 28, 1995·Journal of Chromatography. a·P J Wirth, A Romano

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