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...

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Bibliographic Details
Published in:Proteomics (Weinheim) 2009-05, Vol.9 (9), p.2494-2502
Main Authors: Huang, Kuan Yen, Filarsky, Michael, Padula, Matthew P, Raftery, Mark J, Herbert, Ben R, Wilkins, Marc R
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Blue native PAGE
Chemical Fractionation - methods
Complexome
Continuous elution electrophoresis
Electrophoresis, Polyacrylamide Gel - methods
Fractionation
Fundamental and applied biological sciences. Psychology
Mass Spectrometry
Miscellaneous
Molecular Weight
Multiprotein Complexes - analysis
Multiprotein Complexes - chemistry
Proteins
Proteins - analysis
Proteins - chemistry
Proteomics - methods
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins - analysis
Saccharomyces cerevisiae Proteins - chemistry
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Summary: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.
ISSN:1615-9853
1615-9861
DOI:10.1002/pmic.200800525