Micropreparative fractionation of the complexome by blue native continuous elution electrophoresis.
The large-scale analysis of protein complexes is an emerging challenge in the field of proteomics. Currently, there are few methods available for the fractionation of protein complexes that are compatible with downstream proteomic techniques. Here, we describe the technique of blue native continuous elution electrophoresis (BN-CEE). It combines the features of blue native PAGE (BN-PAGE) and continuous elution electrophoresis (CEE), generating liquid-phase fractions of protein complexes of up to 800 kDa. The resulting complexes can be further analysed by BN-PAGE, by SDS-PAGE and/or by MS. This can help define the constituent proteins of many complexes and their stoichiometry. As BN-CEE is also micropreparative, with a capacity to separate milligram quantities of protein complexes, it will assist the study of proteins of lower abundance. In this regard, the acrylamide concentration and elution rate during separation can be controlled to help 'zoom in' on particular high mass regions and thus complexes of interest. We illustrate the utility of the technique in the analysis of Saccharomyces cerevisiae cellular lysate.
Huang
Kuan Yen
KY
Systems Biology Initiative, University of New South Wales, NSW, Australia.
Filarsky
Michael
M
Padula
Matthew P
MP
Raftery
Mark J
MJ
Herbert
Ben R
BR
Wilkins
Marc R
MR