I am basically optimizing a new protocol I developed myself and I need some assistance. I am looking at the amount of OXPHOS supercomplexes in a model with a limited amount of protein in primary cells. By first running a BNPAGE to separate out the native complexes and then denaturing these complexes and running a 2D gel electrophoresis, I should be able to separate out the subunits from these complexes based on weight rather than isoelectric point without having to run multiple samples/ wells.

Using this method I can detect the individual subunits of the OXPHOS supercomplexes. And I can detect complexes I,III and IV easily using abcam antibodies. However I and IV overlap because these antibodies bind to subunits of close molecular weight so I ordered another antibody from abcam with 5 star rating (NDUFB8) which should bind to another subunit about 20 kDa apart but I basically see nothing new besides what I already blotted using this antibody and with a 1:2000 dilution as recommended. Would anyone have recommendations as to why that would occur? All my other antibodies work great using much greater dilutions. Unless there is something wrong with the logic I am using for 2D gels or simply the antibody is outside the sensitivity of the assay despite it having a high rating.