Hi Everyone I am looking for antibodies for ER pathways that works in INS-1 cells or any other cells. There are lots of paper out there but I want to know if anybody have tried these antibodies in INS-1 cells and that has worked perfectly. The antibodies I am interested are as follows:
spliced-XBP1. p-PERK, PERK, p-IRE1 alpha, Total-IRE1 alpha, ATF4, ATF6 active.
Any information will be highly appreciated. Thanks in advance.
Hi Daleep,
of these I have only used total PERK and total IRE from the cell signaling ER-Stress sampler kit and they both worked well. the CS numbers are #5683 and #3294. 1:1000) In general I would go for the CS antibodies- Either in Sampler kits (they have ER-stress and UPR), which are very cheap per AB, and sometimes they offer 40µl (petit) size, which is also reasonable priced.
I haven't used them in INS-1 cells, however, the ABs shouldn't be cell line specific.
Hope that helps. Good luck!
Hi Dr. Muhlenberg,
Thanks for the information. I hope someone put some information about phopho PERK and IRE1 alpha.
i think p-PERK is not that easy, I remember looking for it quite recently. But there are some ABs out there some of them published - you may want to try and find the respective papers on pubmed. If you decide to go for BiP and CHOP as well the Cs Abs from the sampler kit are very good. CHOP is indeed a bit harder to stain - I would go for the first stain on the blot..
Probably you know already but tunicamycin treated cells are a good positive control - 0.1 - 1µM.
@Thomas Mühlenberg
Thanks. I will look into sampler kit. As you said Chop is difficult to stain try the antibody I recommended in my previous post work really good.
@Youtake Oh
Thanks. It will be helpfull if you can add more information about the company.
The Phospho-PERK antibody from Cell Signaling works well in western blotting (catalog #3179). Be sure to include a positive control such as thapsigargin or tunicamycin treatment as many cell lines do not display much ER stress signaling in normal culture conditions.
Hi Daleep,
I've also used anti-XBP1 (M-186) antibody from SantaCruz (sc-7160) successfully. It should be able to detect the spliced and unspliced versions of XBP-1. Here is the datasheet from the website. As Thomas Mühlenberg and Tom Cunningham mentioned, using either tunicamycin or thapsigargin (Sigma-Aldrich) treated cells as positive controls would be crucial to this experiment. There is also a temporal aspect of the UPR associated with signaling from the three branches, in addition to differences between acute and chronic ER stress in a cell-specific manner, that might also need some optimization.
Hope this helps, good luck!
Viraj
the three most used markers for ER stress: CHOP, ATF4 and ATF6.
Thanks guys for your inputs. Unfortunately couldn't get good results with P-PERK and IRE1 alpha.
Hi Daleep,
do you have a positive control, i.e. a cell line which surely expresses (or is inducible for) pPERK and IRE1? also as mentioned above this might be time sensitive, so after 6h,24h and 48h you may observe different things.
UPR/ER stress is activated through an orchestrated interplay of signaling molecules from all three arms of the UPR pathway i.e. IRE1, ATF6, and PERK. This is why, it is critical to analyze signaling players in the context of the mentioned fact. Novus' ER Stress / UPR Antibody Pack contains sample size vials of the top markers namely: GRP78/HSPA5, pSer724 IRE1 alpha, total IRE1 alpha, XBP1, ATF6, and CHOP/GADD153. For scientific technical answers to over 15 similar questions, I would suggest reviewing our UPR and ER Stress FAQs at https://www.novusbio.com/support/upr-and-er-stress-faqs
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