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BATCH NUMBER 172133 ORDER NUMBER PO104494 DESCRIPTION OF THE PROBLEM I am not getting any signal at the expected size around 80kD. The only thing that comes up in a very long exposure is a weak band at around 66kD. This is not PKC beta as confirmed by blotting with control PKC beta antibody. SAMPLE Mouse B cell extract PRIMARY ANTIBODY used 1:500 in blocking buffer over night at 4 degrees wash in TBS-T DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED that is one of the things I would like to know from you ANTIBODY STORAGE CONDITIONS I stored it at -20 upon arrival, however, it was at room temperature when it arrived, because it was not shipped on dry ice. SAMPLE PREPARATION cells lyzed in buffer containing 150mM NaCl, 50mM Tris, 1mM EDTA, 10% glycerol, 2%ODG (detergent) SDS-sample-buffer containing 2-Mercaptoethanol added and sample boiled prior to gel-loading AMOUNT OF PROTEIN LOADED 30ug ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE 9% TRANSFER AND BLOCKING CONDITIONS Tris-glycine buffer, semi-dry blotting 2h, transfer confirmed by Ponceau-stain block in 5%milk in TBS-T SECONDARY ANTIBODY anti-rabbit-HRP 1h RT(worked well on other membrane developed in parallel) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES Could you please tell the specific conditions how you have used the antibody (how much protein on gel, what dilution, buffer etc). Also, is there something that I can use as apositive control to make sure that the antibody actually works? I am a bit concerned, because it arrived at room temperature. Thank you, Alina
Asked on Jun 27 2006
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.
Answered on Jun 29 2006