For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
If you continue without changing your cookie settings, we'll assume you’re happy with this.
BATCH NUMBER 202129 ORDER NUMBER 182689 DESCRIPTION OF THE PROBLEM no band SAMPLE 3T3 cell lysate PRIMARY ANTIBODY ab1427 1µg/ml in blocker for 1-2 hours, wash 3x5min in PBS DETECTION METHOD Fluorescence on a kodak IS4000MM using proper excitation and emission filters for the secondary antibodies POSITIVE AND NEGATIVE CONTROLS USED all samples were positive controls. ANTIBODY STORAGE CONDITIONS -20 degrees SAMPLE PREPARATION reduced and boiled 5min @95 degrees AMOUNT OF PROTEIN LOADED 20µg ELECTROPHORESIS/GEL CONDITIONS reducing 4-12% bis-tris gel 125V for 2 hours TRANSFER AND BLOCKING CONDITIONS overnight at 30V 5% milk + PBS blocker SECONDARY ANTIBODY Goat anti-rabbit Qdot 705 and Goat Anti-rabbit Alexa 750 at 1:1000 in blocker for 1 hour, wash 3x5min in PBS HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Changed the cell lysate to A431 cell lysate, no change
Asked on Nov 08 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 questionnaire and I have a few comments. You are largely following an approach that I would recommend; using 3T3 mouse cells, a cell type that has been routinely used in the literature as a positive control for the detection of Hsc70 by western blotting. Can you tell me whether you have performed a ponceau red detection of the transfered protein or confirmed the integrity of the lysates using an alternative antibody that targets a "housekeeping" protein such as GAPDH. In the lab at Abcam we frequently find that a change to the blocking agent and/or antibody diluent produces more specific sensitive results. On this occasion I would like to recommend that you try applying the antibody using 3% BSA as a blocking agent, replacing milk. I would also like to recommend the use of TBST (as opposed to PBS) as the antibody diluent and wash buffer. Should a change to the blocking agent not improve your results please get back in touch with me and I will be happy to offer you a replacement vial (provided that the purchase was made within the past 90 days). I hope this information helps, please do not hesitate to contact us if you need any more advice or information.
Answered on Nov 09 2006