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BATCH NUMBER 135821 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM High background, Nonspecific band He got also a specific band at the size of 120kDa. And he found white precipitation in antibody solution. SAMPLE DND39 cells, human PRIMARY ANTIBODY cat.no.ab412, 1:5000, overnight at 4 C DETECTION METHOD HRP-based method POSITIVE AND NEGATIVE CONTROLS USED N/A ANTIBODY STORAGE CONDITIONS One of our customer has stored it at 4 C. We received it on 9th Nov. 2005 and distributed it on 15th Nov. 2005. Cosmo stored it at -20 C for 7 days. SAMPLE PREPARATION Buffer; 50mM Tris, 150mM NacL, 1mM EDTA, 50mM NaF and 30mM Na4P2O7 Protease inhibitor; 1mM PMSF, 2ug/mL aprotinin, 1mM perranadate MATRIX USED Protein A agarose Dilution of primary antibody; 1:5000, overnight at 4 C AMOUNT OF PROTEIN LOADED The number of cells; 2 X 10E6 ELECTROPHORESIS/GEL CONDITIONS Reducing, 8% acrylamide gel, nitrocellulose membrane SECONDARY ANTIBODY [competitor], HRP-conjugated goat anti mouse IgG antibody, 1:10000 His secondary antibody is working well using other primary antibodies. ADDITIONAL NOTES He had purchased lot number 75352, but he got the same result (high background) as this time. Could you please advise us to improve his results.
Asked on Feb 15 2006
Thank you for your enquiry. I am sorry to hear that your customer has been having difficulties with this antibody. I have read their technical questionnaire and I have a few questions. Please can you ask your customer where they consider the problem to lie. I consider that it could be in several places. They may be getting non-specific background during the immunoprecipitation process which could be attributable to the preparation of the lysate or the protocol itself; wash conditions may not be stringent enough. I would like to recommend that the lysate is prepared in RIPA buffer and our standard IP protocol is performed: https://www.abcam.com/assets/pdf/protocols/Immunoprecipitation%20protocol%20(IP).pdf The western blot detection could be part of the problem. I would like to recommend that BSA is used as the blocking solution rather than Milk as we consistently find that this gives cleaner, more specific blots. Finally I would like to remind your customer that this antibody was raised using Asymmetrical NG/NG-dimethyl arginine and will detect any proteins with methylated Arginine. Therefore this antibody will detect several proteins from a whole cell or nuclear lysate. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.
Answered on Feb 17 2006