Gel polymerization in detergents: Conversion efficiency of methylene blue vs. persulfate catalysis, as investigated by capillary zone electrophoresis

Abstract

Four types of detergents are commonly used in biochemical analysis of proteins and polypeptides: neutral, e.g., Triton X-100, Nonidet P-40; anionic, typically sodium dodecyl sulfate (SDS); cationic, e.g., cetyltrimethylammonium bromide CTAB), and zwitterionic, e.g., sulfobetaine 3-12 and 3[(3-cholamido-propyl) dimethyl-ammonio]-1-propane sulfonate (CHAPS). These detergents are utilized not only in the protein solubilization step, but also in the polyacrylamide gel matrix in which subsequent electrophoretic separation is carried out. The conversion efficiency of monomers into the growing polymer, in the presence of the four types of detergents, was assessed by capillary zone electrophoresis (CZE) in a micellar system comprising 100 mM SDS, by extracting unreacted monomers from the gel phase and quantifying the peaks separated by CZE. Two different catalyst systems were evaluated: the standard persulfate-N,N,N',N'-tetramethylethylenediamine (TEMED) couple and a mixture comprising methylene blue in presence of the redox couple sodium toluene sulfinate and diphenyliodonium chloride. In the chemically initiated system (persulfate), there was a strong inhibition of polymerization, decreasing in the following order: CTAB > sulfobetaine 3-12 > SDS > CHAPS > Triton X-100 (e.g., in 10 mM CTAB 100% inhibition was experienced). On the contrary, in photopolymerization (as driven by methylene blue) good conversion of monomers into the growing polymer (> 95%) was always obtained, independent of the nature of the detergent present in the polymerization step.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction and alkylation of proteins in preparation of two-dimensional map analysis: Why, when, and how?

“…It should be borne in mind, in fact, that, whereas the risk of acrylamide adduct formation is much reduced in IPG gels (but not in unwashed IEF gels! ), it is quite real in SDS-PAGE gels, due to the fact that these gels are not washed and that surfactants, in general, hamper incorporation of monomers into the growing polymer chain [25]. Different alkylating residues on a protein will complicate their recognition by MALDI-TOF analysis, a tool much in use today in proteomics.…”

Current challenges and future applications for protein maps and post‐translational vector maps in proteome projects

“…The elimination of free acrylamide monomer in polyacrylamide will be a priority, and may necessitate the use of an alternative polymerisation system such as methylene blue. This is reported to achieve higher polymerisation rates than persulfate catalysis in the presence or absence of SDS [73]. Alternatively, reagents which quench the double bond of the reactive acrylamide monomer could be incorporated into gels [5].…”

Extended investigation of tube-gel sample preparation: a versatile and simple choice for high throughput quantitative proteomics

“…Acidic SDS samples were first brought back to neutrality by adding an identical volume of Tris 100 mM to the sample volume. CTAC-containing samples cannot be polymerized by this method irrespective of the pH because of the CTAC-persulfate precipitation 18 , which leads to polymerization inhibition 22 .…”