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DESCRIPTION OF THE PROBLEM wrong band size ,more bands, SAMPLE human tissure PRIMARY ANTIBODY ab3714 (1:1000;1:500;1:250)dilution:5%nonfat milk/TBS-t=1/4 incubation time:4`covernight ; room temperature 2h;room temperature 1h wash step:TBS-T 10min *2 TBS 10min *1 DETECTION METHOD ECL ANTIBODY STORAGE CONDITIONS subpackage and store -20 dilution store -4 SAMPLE PREPARATION 4*loading buffer ,15ulProtease inhibitors/ml.100`c 5-10min AMOUNT OF PROTEIN LOADED 60ug_100ug ELECTROPHORESIS/GEL CONDITIONS 15% TRANSFER AND BLOCKING CONDITIONS transfer buffer:tris/glycine 300ma 40min (the optimization time verified by ponceau red) blocking :5%nonfat milk 4`c overnight SECONDARY ANTIBODY HRP-antibody from rabbit (1:2000) incubation time(room temperature 2h;room temperature 1h;room temperature 40min) wash step:TBS-T 10min *2 TBS 10min *1 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 15 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES I have got no band when i use ab3714(1:3000) ab3714 lot:164571
Asked on Jul 01 2006
Thank you for your enquiry and for submitting your protocol details. I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionaire in association with your blot images and I would appreciate it if you could address the following questions; 1. Please can you confirm that the samples that you are running are tissue samples. 2. Can you tell me how you are preparing the tissue lysates; the details in the questionaire were confusing. What buffer are you using. 3. In an earlier email you mentioned that "The 29KD lane in the PVDF when is dealt with ponceau red". Does this meant that the marker lane in the blots that you emailed me has a molecular weight marker of 29KDa. 4. Can you tell me the percentage of Tween in the TBST that you are using. You mention "TBS-t=1/4". Can you please tell me the percentage of Tween 20 that you have been using. I ask because there is noticeable noise in the immunoblots that you have emailed me. 5. Can you tell me whether you have been able to obtain good results using this approach with an alternative antibody but the same tissue lysate. This would confirm that the approach, reagents, secondary antibody, transfer conditions etc that you are using are ok. I appreciate your patience and look forward to hearing from you.
Answered on Jul 04 2006