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Micropreparative one‐ and two‐dimensional electrophoresis: Improvement with new photopolymerization systems
Author(s) -
Rabilloud Thierry,
Vincon Mathilde,
Garin Jerome
Publication year - 1995
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.11501601234
Subject(s) - persulfate , methylene blue , polymerization , potassium persulfate , chemistry , polyacrylamide , chromatography , toluene , riboflavin , electrophoresis , photoinitiator , polymer chemistry , organic chemistry , polymer , monomer , biochemistry , catalysis , photocatalysis
Abstract To improve the efficiency of one‐ and two‐dimensional electrophoresis for micropreparative purposes, the use of gels polymerized with other initiators than the standard N , N , N ′, N ′‐tetramethylethylenediamine (TEMED)/persulfate systems for sodium dodecyl sulfate electrophoresis has been investigated. We show here that the recently described photoinitiator system, composed of methylene blue, toluene sulfinate and diphenyliodonium chloride, leads to a decreased resolution. Resolution can be restored if methylene blue is replaced by riboflavin. Two‐dimensional electrophoresis with mg loadings of proteins has also been evaluated with these systems. Independently of the polymerization system, resolution for the first dimension is low with rod gels, increases with gel strips and is further improved when immobilized pH gradients are used. Here too, only the riboflavin/sulfinate/iodonium system results in a resolution that matches the one obtained with the standard TEMED/persulfate system. Gels polymerized with the riboflavin/sulfinate/iodonium system yield better results upon N ‐terminal microsequencing after blotting than gels polymerized with the standard TEMED/persulfate system.