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ANTIBODY CODE ab2914 BATCH NUMBER 28002 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal at the 45-55 kDa range- no bands whatsover in this range. Non specific band at 70 and 30 kDa. MW markers known to be good. SAMPLE We have tested 3 separate blots. The first was with Mouse brain tissue homogenates in RIPA (NP40, Deoxycholate). The second was with mouse brain tissue homogenates in RIPA (SDS). The third was from an IP from mouse brain previously blotted for Barrestin1 (in house antibody, very selective for Barr1- band was there- Abcam antibody did not detect it). On one of the blots, lysates from HEK-293 cells were also included. Barr1 AB detects this human form- the Abcam AB did not detect it again. PRIMARY ANTIBODY We have tested 1:500, 1:250 and 1:100 dilutions of the AB- SECONDARY ANTIBODY The secondary AB has been used with the other Barr1 ab successfully. We use a 1:2000 dilution, 1 hour- anti Rabbit Jackson immunoresearch labs DETECTION METHOD ecl- Pierce- This all works for the other Barrestin AB- POSITIVE AND NEGATIVE CONTROLS USED The other Barr1 AB detects the ~48 KDa band- nothing is present with the Abcam antibody on the same blots or on parallel blots. We really want to detect Barr2 as well- we hoped the Abcam AB would be useful for looking at both proteins simultaneously. However, we cannot detect either. ANTIBODY STORAGE CONDITIONS 4C- just received last week SAMPLE PREPARATION Brain regions or Cell lysates.- the real control is that we are able to detect Barr1 using another antibody on these same blots. It's not our tissue, blotting conditions, etc. AMOUNT OF PROTEIN LOADED We have tested 10 ug to 100 ug protein loaded. 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? protein levels, dilutions, incubation times (o/n 4 degrees C at 1:100- still nothing.) ADDITIONAL NOTES The Barrestin ABs are notoriously nonspecific. we were hoping to be able to determine Barr1 vs. Barr2 via MW (which is possible). However- this antibody recognizes neither form in mouse or in human. It is odd considering the epitope is preserved in these species. Further, a comment on your website suggested it was good for human- Could we have a bad batch? Any assistance would be invaluable. Having a good Barrestin antibody would benefit more than ourselves.
Asked on Mar 09 2004
I am sorry to hear that you are experiencing trouble with this antibody. I have enquired with the originator of ab2914 and everything appears fine with your protocol. We have had no other complaints about this antibody, but this particular antibody has not yet been tested with mouse so maybe it doesn't work in mouse.
Answered on Mar 11 2004