An agarose-acrylamide composite native gel system suitable for separating ultra-large protein complexes.
An agarose-acrylamide composite native gel (CNG) system has been developed for separating protein complexes of ultra-large molecular sizes (over 500kDa) and for analyzing protein-protein interactions in their native states. Various native gel conditions were explored and techniques were improved to facilitate the formation and performance of the CNG system. We demonstrate here that the CNG technique is capable of resolving a complex of RNA polymerase II and an associated factor from the free components, which had not been previously achieved with other methods. Furthermore, this CNG electrophoresis can be conveniently coupled to second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identification of protein components within discrete complexes separated during the CNG run. The CNG technique is particularly suitable for capturing dynamic protein-protein interactions as exemplified here by the formation and demonstration of RNA polymerase II-Fcp1 complex.
Suh
Man-Hee
MH
Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA. mhs35@cornell.edu
Ye
Ping
P
Datta
Ajit B
AB
Zhang
Mincheng
M
Fu
Jianhua
J
D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000378:metabolism
D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D012319:RNA Polymerase II; D010749:Phosphoprotein Phosphatases; C116022:carboxy-terminal domain phosphatase