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one more question about ab31369-- what does it specifically recognize near the C terminus? could it recognize STAT1 that is cleaved at aspartic acid 694? (lower band in earlier attachment seems unlikely to be STAT1 beta since it may not respond to IFNg at all)
Thank you for the update. I had a few more questions and would appreciate any information you can provide.
ab31369:Does this detect total STAT1 (alpha and beta forms, and more importantly, with or without phosphorylation?)
ab30645: Does this only detect STAT1pY701 and definitely not STAT1pS727? What about STAT1 phosphorylated at both sites?
I've attached some blots, including the image below (STAT1 +/- phosphorylation bands range from ˜85 to 100kDa).Is it possible that the:
top bands are STAT1 alpha phosphorylated at Y701
middle (faint) bands are some sort of an isoform
lower bands are either 1) STAT1 beta (or alpha (less likely due to MW?)) phosphorylated at only S727 or 2) STAT1 alpha phosphorylated at both Y701 and S727?
ab11909: Under certain protein isolation conditions, is it possible to lose inactive ATF6 and only detect cleaved ATF6? What would those conditions be and how would the full-length ATF6 be lost?
Please let me know if you have any questions. Thanks much for your help in advance.
Asked on May 07 2012
Thank you for your emails and for your patience while I've looked into these questions.
I've heard back from the lab regarding the specificity of ab31369. The antibody should detect both the alpha and beta forms, thoughwe haven't specifically tested whether it reacts with the phosphorylated protein too. There might be some reactivity with the phosphoprotein, but we would expect it to be weaker than reactivity with the non-phosphorylated protein. Based on our conversation last week and the images you sent, it looks like the antibody is reacting quite strongly with the phosphoprotein. The lab might be running some tests to determine this, and I'll keep you posted on anything else that we find out. I'll also need to check whether the antibody could detect STAT1 cleaved at residue 694, as I don't have the full immunogen seequence in my notes.
Ab30645 is specific for STAT1 phosphorylated at Y701, and I would expect it to react with protein phosphorylated at both Y701 and S727. Some phospho-specific antibodies show weak cross-reactivity for other phospho-proteins, so it is possible that ab30645 would weakly cross-react with protein only phosphorylated at S727. I'm not sure that this has been specifically tested by the lab, but I'll check with them and get back to shortly with that information.
Regarding the images, based on molecular weights I would expect the higher band to be the alpha isoform phosphorylated at both Y701 and S727, while the lower bands would be either the beta isoform (phosphorylated or not) or alpha isoform only phosphorylated at Y701. Phosphorylations eachtend to add some weight to the protein. I think further studies might need to be done in order to know this for sure, but that seems to be the most likely explanation. There are too many possible post-translational statesat similar molecular weights in order to know for sure what each band represents based on band size.
For ab11909, I may not be understanding the question as intended, but the full-length inactive ATF6 protein is cleaved by proteases during ER stress, so I would expect this form of the protein if the cells are under this type of stress. The full-length ATF6 most likely isn't completely lost, butthe cleaved form could certainly be more abundant in the sample and the inactive ATF6 at undetectable levels. It is also possible for proteases in the samples to cleave the protein during protein isolation/sample preparation but this would generally result in several lower molecular weight bands on the Western blot. If the samples are not treated with enough protease inhibitors (or the right mix of protease inhibitors) or were not kept on ice during preparation, it is possible for this to occur. If the samples were treated correctly, and the cells were under ER stress, than I expectthat specific proteases just cleaved the protein to yield the active form. Please let me know if I misunderstood the question or if there is more information that you're seeking.
I hope this information will be useful, but please let me know if you have further questions or if there is anything else that we can do for you. I'll be back in touch once I have more information from the lab about the STAT1 antibodies.
Answered on May 07 2012