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I recently purchased a GPR30 antibody (ab39742) from Abcam and have been having some difficulty optimising it for western blotting. The main problem has been the appearance of non-specific bands (see picture).
I have been using rat brain as a control (lanes 2, 7 and 12) which produce 2 bands relatively close to each other. The other lanes represent human non-pregnant myometrium and appear to produce bands at around 40-45kDa and 70kDa, with a faint one around 130kDa. I was just wondering if this was a common finding and whether I could reduce these non-specific bands? I'm presuming that the lower one is the desired band? The 70kDa band appears stronger, however.
I blocked each membrane for 1hr at room temp in 5% milk.
Primary antibody was incubated in 1 % milk overnight at 4 degrees.
Secondary antibody (diluted to 1:3000) was also incubated in 1% milk.
Milk was diluted in TBS-Tween.
PVDF membranes were used for the blots.
I was just wondering if you had any advice, or know of any similar issues with other clients?
Asked on Jan 30 2012
Thank you for bringing this to our attention.
We have received several complaints over the past two years but without consistent observations, and none similar to yours. Our own data has the protein appearing at about 52 kDa. The assumption has been that glycosylations slow down migration in the gel. The predicted molecular weight of 42 kDa is based on the primary amino acid sequence, before modification post-translation.
So it is difficult to confidently say that the bands near 45 kDa in your blots are in fact GPR30 or are instead another protein that the antibody cross-reacts with. For the rat brain samples, a smear rather than two distinct bands is more expected, as a consequence of different amounts of glycosylation. We have not seen a band at 70 kDa before.
Can you please tell me how the samples were prepared before loading in to the gel? Also, how much protein was loaded per lane? Have you staining blots of these samples for any other proteins, and if so, were the results as expected?
Can you please send the order number or approximate date of the order? If I cannot make a suggestion, we have another lot available which may be worth a try.
Answered on Jan 30 2012