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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM For the TRPM8 a size of about 130 kDa is predicted for human and also rat protein. However Xiao-Ru Yang et al found a band at 250 kDa as TRPM8 (see attachment). What do you think about these data and which size do you predict? I already tried tongue and bladder of rat as positive control. Unfortunately I find several unspecific bands. Does abcam provide an adequate positive control in order to find out it the signal comes at 250 or 130 kDa? Thank you very much for your help. SAMPLE Rat, adrenal medulla, ductus deferens PRIMARY ANTIBODY Abcam Anti TRPM8 POSITIVE AND NEGATIVE CONTROLS USED Positive control: tongue, bladder of rat ANTIBODY STORAGE CONDITIONS -20°C dissolved in BSA/TBST/NaN3 --> 4°C SAMPLE PREPARATION Preparation with TRIS/SDS, heating at 95°C, 5 min. Then loading buffer plus protein again heated at 95°C for 5 min. AMOUNT OF PROTEIN LOADED 70 µg ELECTROPHORESIS/GEL CONDITIONS 30:0,8 Acrylamide 8% TRANSFER AND BLOCKING CONDITIONS Blocking agent:5% milk/TBST (BSA not yet tryed) overnight SECONDARY ANTIBODY non Abcam, Peroxidase-conjugated Goat AntiRabbit
Asked on Aug 07 2007
Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab3243 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 can confirm that our laboratory tested this antibody with transfected Cos cell lysate in western blotting and managed to obtain a band at around 130kDa. Unfortunately, I am unable to retrieve any further protocol information (amount of protein loaded, etc.) and therefore, the image is not suitable for use on our datasheet. I have attached a copy of the image for your reference only. I am unsure of the exact protocols used by Yang et al 2006, but please rest assured that we guarantee our products to perform well in species and applications as stated on the datasheet. If you would like some technical support, I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. 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. What was the dilution that you used? 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) and secondary antibody concentration or run a no-primary control (without the primary antibody). Running a no-primary control will be able to eliminate the possibility that your secondary antibody is binding non-specifically. Another possible cause of non-specific bands is that the amount of protein is too much. We normally recommend using 20-40 micrograms per well. Please try this amount if you have not already done so. 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 rather than 5 minutes to ensure proper disruption of multimers. 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. Increasing the washing frequency and duration would also help eliminate non-specific bands. Can you confirm that you have used Tween in your washing solution and have washed at least 15min x 3 times? 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 info, contact information, etc.) 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. I look forward to hearing from you again in order to resolve the matter.
Answered on Aug 07 2007