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Inquiry: I purchased ab56280 c-Fos protein (Tagged) to use as a substrate in a kinase assay - as listed under functional studies on your product description page. Unfortunately this product is already phosphorylated on T232 (detected using your antibody (ab43175)) which makes it unusable in a kinase assay. I ran the protein alone on a western blot and it had a very high signal for phospho T232. I ran 0.08 µg of C-fos protein and used 1:2000 dilution O/N for primary and 1:6000 for 1h for secondary. Blocked in milk-tbst.
Asked on Feb 28 2013
Thank you for contacting us.
We produce c-FOS in insect cells, so the protein may have some phosphorylation levels in some sites. We’ve tried to limit the kinase activity during the production process, including the expression stages and purification steps, but we can’t protect its phosphorylation completely. Also, we can’t test the modification levels in all of the sites, but we still think our c-FOS protein is a good substrate for most of application assays. Even you had the positive signal in the Thr232, we don’t think that site has been fully phosphorylated, and you may have used too much protein (˜80ng) to do the WB.
We would suggest that you do a dilution study (such as starts at 20ng>>>) in WB with phosphor-antibody and protein-antibody, and to see if you can get the protein amount point which shows the positive signal in protein-Ab and negative one in phosphor-Ab. You may then use the optimized protein amount to do his test
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Answered on Feb 28 2013