ECM1 on a western blot, why is one band much lower than the rest? - (Nov/09/2015 )
I ran a WB looking at expression of ECM1 in several cell lines. The results were as expected, however for one of the cell lines the band was significantly lower (around 60-65 kDa) than for the other cell lines (all of which showed a band at around 85 kDa). I was using a Santa-Cruz ECM1 antibody, N-17. Has anyone else experienced anything similar, or know why this might be. I wondered if it either this cell line was expressing a different isoform of the protein (ie ECM1b) or if the protein was less glycosylated..?
There could be a number of reasons:
Western blot based: non-specific binding... single bands don't necessarily indicate specific binding. Sample buffer composition different for that sample,
Cell based: DNA sequence mutation/truncation, RNA truncation, protein mis-translation or modifications not present (e.g. cell isn;t capable of full glycosylation), isoform detected, protein not folded correctly.