I have a western blot which I incubated with an antibody anti-ADH 7.
I had a faint band appearing at approx 42 kDa. When I incubated with anti-actin afterwards, a band at approx 42 kDa appeared, with the same size and same shape of the band that had appeared prior to incubating with the actin antibody.
Could my antibody be cross-reacting to actin? Could this just a coincidence and they are just different bands with the same weight and same size?
Hello,
The first thing I would do is contact the manufacturer of the primary if the expected molecular weight of your target protein is not 42 kDa. However, a quick search for anti-ADH7 primary antibodies shows reactive bands are common at 41kDa. Being that Actin and potentially ADH7 have such a close MW, if it were me I would use a different loading control, perhaps probe for alpha or beta tubulin, which have a MW of about 55 kDa.
Hi Diogo, could you provide more detail.
Was this 42K band visible from Ponceau staining of the blot prior to ab staining?
As Adam said, the antibody manufacturer might be bad.
You could also try to run native PAGE of your sample and blot. The ADH dimer should show up.
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