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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Non-specific band, and high background SAMPLE Cell extract PRIMARY ANTIBODY Abacm ab32815, tried 1:1000, 1:500 in TBST with 1%milk cold room, overnight DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED we have the cells tested by RT-PCR for Igf2r expression, also have knock-out cells as negative which were confirmed by RT-PCR ANTIBODY STORAGE CONDITIONS -20 C after receive it SAMPLE PREPARATION RIPA buffer lysis, plus protease inhibitors (Pierce Cat 78410) AMOUNT OF PROTEIN LOADED 25 ug per line ELECTROPHORESIS/GEL CONDITIONS Pierce Precise protein gel (4-20%) TRANSFER AND BLOCKING CONDITIONS Tris-Glycine transfer buffer, Blocking in TBST with 5% milk, room tempeture for 1.5hr SECONDARY ANTIBODY Jackson ImmunoResearch 711-035-152 1:10,000 dilution work well in the lab HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? last step after got the results ADDITIONAL NOTES many unspecific bands on the film, no difference between positive and negative samples PO number for this: RSTFD0000209430
Asked on Sep 04 2007
Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab32815 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. What species of cell extract are you using? 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. You can try decreasing the primary (1:2000 ~ 1:5000). Also, running a no-primary control experiment will be able to eliminate the possibility that your secondary antibody is binding non-specifically. 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. If you can provide an image, that would certainly help me understand the problem better. Since you mentioned that you seeing high background, I would assume that it is the milk that is causing the problem. In general, there are two blocking solutions that are widely used, non-fat milk and BSA. We would recommend using 5% BSA as almost all of our products are tested with it and have proven to be effective. Most customers who use milk usually have high background, due to the secondary antibody binding to the milk. You can check if this dark signal you are detecting is from the secondary or not by incubating your blot in secondary only. Increasing the washing frequency and duration would also help eliminate high background. Can you confirm that you have used Tween in your washing solution and have washed at least 15min x 3 times? Please try this if you have not already done so. I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with the answers to the above and your shipping address. Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.
Answered on Sep 04 2007