I am dealing with a predicted 40 kDa MAP kinase in WB. I don't have access to LC/MS to make sure of its MW, so I rely on that of the strongest band detected (I'm not worried about faint ones because my MAbs are highly specific). But what I found doubtful is the following:
(i) The major band corresponding to the phosphorylated form is always ~15 kDa heavier than its nonphosphorylated counterpart (37 kDa).
(ii) I still get a second matching band (that of 37 kDa), but with a much weaker signal.
So could phosphorylation or whatever other kinds of PTMs shift up the MW that far? Or shall I worry about the antibodies specificity?
I suggest you always worry about antibody specificity. Check, for example, the article:
Uhlen M et al, Nature Methods volume13, p.823–827 (2016)
Phosphorylation can shift the apparent molecular weight significantly: so much, that replacing a single Tyr site by Phe in the almost 1200 aa full-length EGFR leads to a visible shift of the band of the phosphorylated receptor, while the apparent MW of the unphosphorylated receptor was not affected. The mobility shift is largely due to to reduced SDS binding: https://pdfs.semanticscholar.org/1640/20f32c4b1c7c05d37cb3e300c42a2b5e7315.pdf
If you are worried that phosphorylation may mask your protein from the antibody, you could treat the membrane with phosphatase: see https://www.abcam.com/protocols/protein-dephosphorylation-protocol
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