Anti-PKC beta 1 + PKC beta 2 (phospho T500) 抗体 (ab5817)

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.

Thank you for getting back to me. I have been unable to obtain a satisfactory answer from the source of this antibody. However, I see no reason why the positive control that you have been using would not be suitable. Furthermore the approach that you are using is one I that I would recommend. I am certainly prepared to offer you a credit note against this purchase provided that the antibody was purchased within the past 90 days. If this is the case please email me details of the order including the order number and date of purchase. I look forward to hearing from you.

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionaire and I have a few comments. The approach that you are employing is largely one that I would recommend. I am however concerned that you are not detecting the phosphorylated form of PKC beta given as a result of the low level of phosphorylation of this protein in B cells. We recommend that PMA stimulated K562 cells are used to guarantee high levels of phosphorylation and good signal by western blotting. Unfortunately we do not sell this as a pre-prepared lysate. However, I would like to suggest that a stimulated cell line is incorporated into your experiments to guarantee that you have phosphorylated PKC on your blot for targeting by the antigen. I would also like to recommend that you incorporate protease inhibitors in your lysate preparation as the lack of signal may be the result of protein degradation. The western blotting conditions employed using this antibody are detailed on the antibody datasheet: Membranes were blocked with 5% BSA-TBST and incubated with the antibody diluted in 3% BSA/TBST. Should you continue to experience difficulties please get back in touch with me.

Thank you for your enquiry. This testing has not been performed as far as I know, but with some research I have found the following information: PKC alpha shows ~79% homology within the range of the immunogen sequence. PKC beta I shows 50% homology within the range of the immunogen sequence. PKC delta shows ~18% homology within the range of the immunogen sequence. PKC epsilon shows 50% homology within the range of the immunogen sequence. PKC gamma shows ~64% homology within the range of the immunogen sequence. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Thank you for your enquiry. Are you interested in detecting PKC beta 1 or 2? Ab5785 detects PKC beta 2 phosphorylated at T641, where as ab5817 detects PKC beta 1 and 2 phosphorylated at T500. For both of these antibodies there are Western blot images on the online datasheets, and we guarantee that the antibodies will work in Western blotting with human samples. I hope this helps. If you have any additional questions, please contact us again.

Thank you for your enquiry. Useful working concentration range of PMA is 1–100 nM. You may need to consider testing the half-maximal effective dose of PMA in your experimental system. Please take a look at the following website, you may find some useful information on PMA and effective concentration: http://www.promega.com/cnotes/cn001/cn001_10.pdf