Micropreparative one- and two-dimensional electrophoresis: improvement with new photopolymerization systems.
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.
CEA, Laboratoire de Biologie Moléculaire du Cycle Cellulaire, INSERM U309, DBMS/BMCC, CEN-G, Grenoble, France.
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Rabilloud
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Vincon
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Garin
D000645:Ammonium Sulfate; D001713:Biphenyl Compounds; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005029:Ethylenediamines; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D007782:Lactoglobulins / Q000737:chemistry; D008027:Light; D009861:Onium Compounds; D010777:Photochemistry; D011108:Polymers / Q000737:chemistry*; D012256:Riboflavin; D017421:Sequence Analysis; D013441:Sulfinic Acids
D001713:Biphenyl Compounds; D005029:Ethylenediamines; D007202:Indicators and Reagents; D007782:Lactoglobulins; D009861:Onium Compounds; D011108:Polymers; D013441:Sulfinic Acids; C031291:diphenyliodonium; C005798:N,N,N',N'-tetramethylethylenediamine; C031276:ammonium peroxydisulfate; D000645:Ammonium Sulfate; D012256:Riboflavin