Question (14674) | Anti-pan Arrestin antibody (ab2914)

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Question

BATCH NUMBER 118458 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM wrong band size - expected 47/49 kDa, but only two strong bands at ~83 and 40 kDa, and a few fainter bands, but none of the expected size. SAMPLE cell lysates - SK-N-MC, H295R PRIMARY ANTIBODY primary antibody used 1:333 in milk in TBS tween, incubated overnight at 4 degrees C with agitation. blots were washed 3 times at room temperature with agitation for 10-20 min per wash. DETECTION METHOD ECLPlus ANTIBODY STORAGE CONDITIONS reconstituted in water, aliquots stored in -20 degrees C SAMPLE PREPARATION protease inhibitor added, samples boiled for 5 min in SDS lysis buffer AMOUNT OF PROTEIN LOADED 50 ug ELECTROPHORESIS/GEL CONDITIONS 10% reducing gel TRANSFER AND BLOCKING CONDITIONS blocked with milk in TBS tween for 1 hour at room temperature, with agitation SECONDARY ANTIBODY 1:5000 in milk in TBS tween, incubated for 1 hour at room temperature. blots were washed 3 times at room temperature with agitation for 10-20 min per wash. 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

Answer

Thank you very much for your protocol details, I think there may be a step which will also help you (as well as running the positive controls mentioned in my previous e-mail-brain and spleen). In our experience multiple non specific bands can be caused by too much blocking as the milk causes the antibody to bind to lots of proteins. I would therefore recommend trying: -a different blocking agent: I have very good experience of 5%BSA in TBST (1hr, RT) and incubating the antibody in TBST only -less milk: try 30min incubation in 5%milk then rinse gently in TBST and incubate the antibody in TBST only at 4C. From the papers I have read about arrestin the protein is found in the cytoplasm so you should not need a very strong detergent to extract it, however I would recommend checking that the lysis buffer is fresh and the protease inhibitors too and samples are kept on ice at all times. Finally can I make sure the loading buffer contains reducing and denaturing agents? I hope the above advice will help you, please do not hesitate to contact me again if you need further assistance,

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