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Enclosed please find the western blot result by using ab3243. The exprimental condictions are as follows: 1. Loading total protein 40ug/lane in 9% separation SDS-PAGE gel (The second lane: bladder, as my postive control). 2. transfer to PVDF membrane (Millpore immobilon-FL), 47 Volt for 60 min. 3. blocking: 5% skim milk in TBST (0.05% Tween 20) 4. primary antibody: ab3243 (lot: 237021) by using 0.1ug/ml as final concentration at 4oC overnight. 5. secondary antibody: goat anti-rabbit HRP (Pierce) 1:2500 dilution at room temperature for 1 hour. 6. ECL: Supersignal west Pico Chemiluminescent (Pierce 34080)
Asked on Jan 26 2007
Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionaire and I have a few comments. We have details of this antibody applied by IHC from customer feedback, but not western blotting. You are obtaining an alarming number of extraneous bands. I would like to recommend that in order to obtain more specific results that you use 3% BSA as a blocking agent. This is routinely employed by our lab team. I would also like to recommend that you increase the percentage of Tween 20 to 0.1% in an effort to address the non-specific nature of this antibody. I would also like to recommend that you perform an overnight block of your membrane using this agent. If this does not improve your results please get back in touch with me and I am happy to provide you with a replacement antibody or credit against your purchase provided that you purchased this antibody within the past 90 days.
Answered on Jan 31 2007