Please, can anyone please help me with this issue? By BN-PAGE and western blot on DDM-solubilized mitochondria, isolated from cultured cells, I found that in treated cells the amount of CV subcomplexes was markedly increased as compared to control cells. However, SDS-PAGE analysis on whole cells (not mitochondria) followed by western blotting using the same antibody did not show any difference. Shouldn't I expect comparable quantitative results between SDS-PAGE and BN-PAGE? Thanks
Dear Paola!
Plase You read these publications:
Article Native SDS-PAGE: High Resolution Electrophoretic Separation ...
Your differences in results are more likely due to the specifics of the methods SDS-PAGE and BN-PAGE:
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used to obtain high resolution separation of complex mixtures of proteins. The method initially denatures the proteins that will undergo electrophoresis. An obvious limitation of SDS-PAGE resides in its deliberate denaturation of proteins prior to electrophoresis. Enzymatic activity, protein binding interactions, detection of protein cofactors, etc. generally cannot be determined on proteins isolated by SDS-PAGE. Instead, other methods must be employed to separate native proteins for investigations of structure-function relationships.1, 5 One such alternative is the blue-native PAGE technique.6 This method has been used in the determination of protein-protein interactions, in which proteins in the sample are separated as oligomers in first dimension BN-PAGE, followed by a denaturing second dimension SDS-PAGE to identify the monomers within the oligomers.7-11 However, as a one-dimensional separation method, it faces the opposite problems of SDS-PAGE. While, BN-PAGE retains the native state of proteins, it falls short of the high resolution of proteomic mixtures that is attained with SDS-PAGE and can add ambiguities to successful molecular weight determinations.12-14
I thank you very much, Alexander. I will read the two publications and try to find an answer to my issue. Thanks again
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