I have ordered another ladder to test, but could use any additional suggestions for solving this problem.Ī Are there any confirmed or predicted glycosylation sites in the human primary amino acid sequence? If so, are those sites conserved in the ferret sequence? It is possible that the protein is processed differently in ferret cells. The prestained marker seems to transfer fine, and Ponceau staining of the membrane shows a reasonable amount of protein throughout the lane. Could either of those treatments result in protein cleavage? Or is it possible that the Kaleidoscope pre-stained marker shows a misleading pattern? I am using 12% SDS, wet transferring overnight at 30V, and performing overnight blots with the primary antibody and non-specific controls while I am troubleshooting the procedure. The samples were denatured for 5 min at 95☌ with beta-mercaptoethanol and then stored for six months at -80☌ or 4 weeks at -20☌. So I'm sure the labeling is coming from the primary antibody rather than from non-specific binding of the secondary antibody. If I pre-incubate the antibody for a negative control, the band vanishes. My problem is that the band is about 40 kDa smaller than expected no band appears at all in the expected position. Each time I do the Western blot, I see a clear, reproducible, concentration-dependent band from each species, which confirms cross-reactivity. I would like to confirm cross-reactivity of the antibody between human, mouse, and ferret. Q I am using Western blotting to look at a membrane channel of approximately 150 kDa in size. Why doesn't my protein band appear at the correct apparent molecular weight? (Thread 19829)
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