p-Smad2 problems in western blot - TGF-beta and p-Smad 2 (Mar/19/2005 )
I'm doing a TGF-beta project. Now I'm assaying for p-Smad2 using western blot. TGF-beta1-stimulated cancer cells should be postive for pSmad-2 on the blot. However I've had problems with my negative controls. All my unstimulated cells are positive for p-Smad2 as well.The technical sheet for my Cell Signalling p-Smad2 antibody showed that their HepG2 assay had very little if not zero basal p-Smad2. I've done assays for HepG2, MDA-MB-231 and MCF-7. No success at the moment. Thank you for your attention
(1) Is the antibody you used been cited by literature?
(2) Is this antibody against p-tyr or p-Ser/Thr or both? Anti-p-Ser/Thr often not so specific.
(3) consider to change another antibody.
(4) The possibility of TGF-b problem - even your control has basal level of p-Smad2, TGF-b should significantly augment the signal. I have problems with growth factor signaling before due to the low potency of the factor. Make sure your TGF-b is from the reliable suppliers like Epoch Biolabs or R&D Systems.