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BATCH NUMBER 148880 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Non-specific band They got an expected band also at the size of 28kDa. But they want to use it for IHC. They were not satisfied with its specificity. So he wants to get a refund. SAMPLE WCL isolated from cultured human TIG cells PRIMARY ANTIBODY 1:100 in 5%BSA/TBS-T, overnight at 4 C DETECTION METHOD HRP-based method POSITIVE AND NEGATIVE CONTROLS USED no ANTIBODY STORAGE CONDITIONS Cosmo received it on 28/Mar./2006 and the researcher received it on 29/Mar./2006. They've stored it at 4 C. SAMPLE PREPARATION 10mM Tris (pH7.5), 2% SDS, 10mM DTT AMOUNT OF PROTEIN LOADED 20ug/lane ELECTROPHORESIS/GEL CONDITIONS 13.5% acrylamide gel, reduced conditions, PVDF membrane TRANSFER AND BLOCKING CONDITIONS 5% skim milk/ 0.5%Tween-TBS at 25 C for i hour SECONDARY ANTIBODY 1:5000 in 5%milk/TBS-T, for 1 hour at room temp. His secondary antibody is working well using other primary antibodies.
Asked on Apr 17 2006
I'm sorry to hear the customer is having a problem with ab24502. I have noticed a few parts of the protocol that may explain why the result is a non-specific band and a specific band. For example, the datasheet expresses states to store the antibody at -20 to -80C. It is entirely possible that the antibody is not stable for the past three weeks at 4 degrees. I would also like to suggest the following modifications to your protocol, however, in hopes that the results will improve: 1) Please be consistent with blocking reagent. I would recommend blocking in 5% milk 1 hr, and incubate in primary and secondary with 5% milk. 2) The primary can be diluted further. 3) Can you clarify the sample type? For example, A1 seems to be restricted to the hematopoietic compartment. It is expressed in peripheral blood, spleen, and bone marrow, at moderate levels in lung, small intestine and testis, at a minimal levels in other tissues. It is also found in vascular smooth muscle cells and hematopoietic malignancies. What are WCL? Whole cultured lymphocytes? 4) Can you please send me a labeled picture of the blot as I will be better able to trouble-shoot with this picture? Please let me know if this helps and do not hesitate to contact us for further advice.
Answered on Apr 19 2006