Question (13165) | Anti-ARH antibody (ab5082)

Go to datasheet (ab5082)

Question

BATCH NUMBER 98818 ORDER NUMBER 90200 DESCRIPTION OF THE PROBLEM Wrong band size detected SAMPLE Number of different tissues: Human fibroblast/ARH null fibroblasts/ Rat liver lysate/Rat brain cytosol/hela cell lysate PRIMARY ANTIBODY Abcam Goat anti ARH (1:500) DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Control fibroblasts +ve control ARH null fibroblast -ve control ANTIBODY STORAGE CONDITIONS -20 degree C SAMPLE PREPARATION As in the standard protocol in the lab AMOUNT OF PROTEIN LOADED ~25 microgram /lane (mini protean from Biorad) ELECTROPHORESIS/GEL CONDITIONS reducing 12.5% SDS Gel TRANSFER AND BLOCKING CONDITIONS 1.15 hours at 110 volts on nitrocellulose tris/glycine SECONDARY ANTIBODY Sigma anti Goat IgG Hrp conjugate 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 WHAT STEPS HAVE YOU ALTERED? None

Answer

I'm sorry to hear you are having a problem with ab5082. Thank you for filling in our online protocol questionnaire, this is very useful to understand your problem and I can already confirm that I looked at your shipping details and there was no delay in transit. We have not tested the antibody in rat tissue but it should work in the positive control of human liver lysate (we have not tried in human fibroblasts or HeLa cell lysate). Could the levels of ARH in your human fibroblasts be below WB detection level? Could you please tell me a little bit more about a few steps in your protocol so I get an even better understanding of your experiment: -what size did you detect? Was it around 40-45kda, below, or above? Did you have other bands suggesting non specific binding? -what is the composition of the lysis buffer? We recommend RIPA buffer with protease inhibitors. I'm sorry for the delay in solving this problem and appreciate you taking the time to give me those last few details to help you, I look forward to hearing from you,

Sign up