I have a problem using Drp1 S637 antibody from cell signaling for western blotting technique. Although it should be at around 70-80kD, I get a band at 120KD. Is this a splice version or a dimer?
We also meet higher brand in DPR1 p637 Ab from cell signal. It follow with the treatment to be stronger or weaker. We think it may be the splicing variant.
The antibody works fine for us. Make sure to treat some of your cells with Forskoline as a positive control for DRP1 phosphorylation at S637. If it shows a big P-DRP1 band so it is the one regardless of their migration size. Also use the total DRP1 antibody from cell signaling to confirm.
I am having the exact same problem with my pDrp1(S637) antibody from CST. What kind of sample are you blotting? And did you figure out why you are seeing the higher molecular weight?
It works well for cell culture of several cell lines, especially if treated with forskoline as a positive control. Animal tissues can be tricky due to isolation procedure. Remember that apoptosis signaling can decrease P-DRP1. Also, muscle and heart tissues have more calcium activity that can affect dephosphrylation rate
I am using a salivary gland tissue lysate, and I am aware of the risks of degradation etc. in preparation. The presence of a higher molecular weight band on an otherwise clean blot is very puzzling to me, though. I cannot fathom why the alleged protein would end up so close to the top of the ladder, unless it's an antibody problem.. My total Drp1 (also CST) blot looks fine, and gives no bands in the upper range.
DRP1 interacts with other proteins and engages in homo-oligomers, but those are hard to catch in normal SDS-PAGE blot. There is always the chance of unspecific binding, especially that the total antibody is not detecting that band
Hi Andre,
I've just used the same antibody looking for S637 p-DRP1 from human MDMs and have got the same size bands as well. The total protein looks fine at around 80 but the phospho-DRP1 gives a band over 100. I'm planning to run some extracts with a cAMP agonist I isolated previously as a positive control but was hoping you had managed to come up with an answer as to whether this is a non-specific band or the genuine protein in a homo-oligomer etc..
Would be grateful for your help!
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