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thank you for your reply. I do use protease- and phosphatase-inhibitors in my lysis buffer, so I don't think, this is the reason that the antibody is not working. Also, I use stimulated B cells in which PKCbeta should be active. Since you don't have a positive control for this product available and I do not have the cell line which you mentioned, can you tell me, if EGF-stimulated A-431 cells would be a suitable control? Thank you
Asked on Jun 29 2006
Further to a follow up email from our source of this antibody I have ascertained that A431 cells are apparently NOT a good choice for the detection of PKC beta. According to them the cells have been shown to express PKC alpha, delta and zeta but not PKC beta. They question the specificity of the antibody that you have been using to detect PKC beta; perhaps it is detecting generic/pan PKC? They suggest that many of the antibodies on the market demonstrate this reactivity. Jurkat or K562 cells are the best positive control cell lines according to their recommendation. There are likely to be many other cell lines in the literature. However, A431 cells are NOT suitable according to their recommendations. Additional info: K562 and Jurkats were treated with PMA, 100 nM for 10 min.; K562 had the higher expression level and better looking signal so were used those for validation purposes. In the absence of any stimulation, one should be able to detect the phosphorylation of PKCbI&II (in response to endogenous stimuli, e.g., cAMP and others). I recommend that he use Jurkat cells treated with PMA as positive control. Let me know if I can assist further. In light of this information I would like to recommend that an alternative positive control is employed. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.
Answered on Jul 11 2006