One‐ and two‐dimensional histone separations in acidic gels: Usefulness of methylene blue‐driven photopolymerization

Abstract

We tested the recently introduced methylene blue-toluene sulfinate-diphenyl-iodonium polymerization system in order to prepare acetic acid-urea-Triton X-100 gels for histone separations. When compared to standard persulfate-based initiators, this system exhibited several advantages. First, the polymerization proceeds at a much faster rate but is easily controlled since it is light-dependent. Second, no prerunning of the gel was required, since there is no oxidizing molecule able to introduce artifacts. Moreover, this procedure produces gels presenting cleaner backgrounds with silver staining. The ability of the procedure to carry out high resolution two-dimensional electrophoresis of histones was also examined. High resolution two-dimensional gels, using photopolymerized acidic gels as the first or second dimension were obtained.

Solubilization of proteins for electrophoretic analyses

“…This system has many desirable properties (reviewed in [86]), including a high polymerization efficiency over a wide range of pH down to pH 3 [77], and insensitivity to the presence of organic solvents or detergents [87, 881. This system was tested for zone electrophoresis in the cationic mode and found to give very good results [89]. Minor problems, however, before cationic detergent electrophoresis becomes as user-friendly as SDS electrophoresis are the absence of good tracking dyes or difficulties in the staining protocols; however, the compatibility of cationic detergent electrophoresis with structural analyses (e.g.…”

Fully denaturing two‐dimensional electrophoresis of membrane proteins: A critical update

“…For example, the polymerization of gels at low pH cannot be achieved by the classical and robust TEMED/persulfate system, and the more delicate ascorbate/ferrous ion/hydrogen peroxide system is often used. Alternatively, the methylene blue-based photopolymerization system [77], which has been shown to be efficient with other types of acidic, urea-containing gels [78], can be used to polymerize the acidic first-dimension gels [79].…”

Cytosolic and Transmembrane Protein Extraction Methods of Breast and Ovarian Cancer Cells: A Comparative Study

“…To validate the efficiency of protein extractions, the total protein lysates were subjected to run in SDS-PAGE for separation of proteins based on their molecular sizes, followed by Coomassie blue and highly sensitive silver staining, which are the usually adopted techniques used in molecular biology. 22,23 We also performed immunoblot analysis using target gene-specific antibodies to identify the low MW cytosolic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and relatively high MW membrane protein integrin b-1 expression in a panel of cancer cell lines. Our studies revealed that Tris-based lysis buffer with 50 mM concentration, pH 7.5, comparatively has better protein extraction efficiency for both cytoplasmic and transmembrane proteins.…”