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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM multiple non-specific bands and band at correct location faint SAMPLE pc3 cell extract PRIMARY ANTIBODY ab53161, overnight at 4 degrees, 1/250 dilution, washed after DETECTION METHOD ecl POSITIVE AND NEGATIVE CONTROLS USED positive control: hep3B, cis-retinoic acid treated cells (make rarB by protein and message), negative control, untreated pc3 cells ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION 4% SDS, roche protease inhibitor cocktail, DTT, nupage lds buffer AMOUNT OF PROTEIN LOADED 25 ug ELECTROPHORESIS/GEL CONDITIONS reducing gel, gradient 4-12% TRANSFER AND BLOCKING CONDITIONS 30V for 2 hrs, blocked in 10% milk in tbst, tbst wash steps SECONDARY ANTIBODY pierce anti-rabbit, 1/5K dilution, incubate 2 hours, washed with tbst HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No ADDITIONAL NOTES similar results with your mouse rarB polyclonal antibody
Asked on Dec 14 2007
Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab53161 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. a) Multiple non-specific bands: What are the band sizes you obtained? The bands located above the expected molecular weight band could be multimers. Please try boiling your protein in SDS-PAGE for 10 minutes to ensure proper disruption of multimers. This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. Meanwhile, bands located below the expected molecular weight may indicate that your target protein has been digested. Please make sure that you incorporated sufficient protease inhibitors and have kept your samples on ice the whole time. In most cases, the cause of multiple bands is because the primary and/or secondary antibody concentration is too high and causes non-specific binding. The dilution that we have on the datasheet is a recommended starting dilution and customers are encouraged to determine the optimal concentration/dilution. Please try decreasing the primary (1:500 ~ 1:3000) antibody concentration. I would also suspect that the secondary antibody is binding non-specifically and causing the extra bands. Please run a no-primary control (without the primary antibody) as this will eliminate the possibility that your secondary antibody is the cause of the problem. b) Weak band at the correct location: Was the lysis buffer you used as strong as RIPA buffer? Please try RIPA buffer as it is the strongest and will ensure that you get all the protein of interest out. I would suggest increasing the amount of protein sample. At Abcam, we recommend using around 20-40ug per well and you may want to try increasing your sample to 40ug per well. Note: a high amount of protein sample may also cause multiple bands. So, please try the troubleshooting tips in section (a) before trying this section. I hope the above recommendations may already help you, if you still experience problems please do not hesitate to contact me with the answers to the above questions and an image of the blot if possible. I would also appreciate if you could also provide details of your order (purchase order number, shipping address/purchasing agent information) so that I can immediately arrange for a replacement or refund to you. Also, please advice on how you would to proceed with this enquiry.
Answered on Dec 14 2007