Question (11985) | Anti-CLLD8/SETDB2 antibody (ab13712)

Go to datasheet (ab13712)

Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 72248 DESCRIPTION OF THE PROBLEM Also purchased ab13712. Multiple bands with each antibody. Molecular weight of bands different for both antibodies. Not sure if I'm picking up the correct band and if so with which antibody SAMPLE Human cell lysates A549, jurkat, C8166, Daudi, HUT-78 PRIMARY ANTIBODY ab5517 or ab13712 SECONDARY ANTIBODY cell signalling anti-Rabbit-HRP #7074 abcam rabbit anti-goat6741-1 DETECTION METHOD chemiglow POSITIVE AND NEGATIVE CONTROLS USED none available SAMPLE PREPARATION RIPA + protease inhibitors, heated 5min TRANSFER AND BLOCKING CONDITIONS NuPAGE blotting buffer + 20% MeOH, 2hr. Blocked 5% milk in PBS-tween HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No WHAT STEPS HAVE YOU ALTERED? primary antibody concentration

Answer

I'm very sorry to hear you are having problems with ab5517 and ab13712. You do not mention adding loading buffer to your samples prior to boiling, please make sure you add loading buffer containing SDS, DTT, beta-mercaptoethanol and bromphenol blue. Please also make sure you keep samples on ice until boiling and that protease inhibitors are added freshly. You do not mention how long you block the membrane for (and what temperature), I recommend trying 5%BSA in TBST for 1 hr, then rinsing a few seconds in TBST and incubating both primary and secondary antibodies in TBST. The dilution recommended for ab5517 is 1:5000 and for ab13712 is ug/ml but you may need to dilute more, I hope these suggestions help, please do not hesitate to contact us if you need further assistance,

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