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To clarify, the conditions I used (which were identical to those used for the [anoter compagny] antibody, appart from primary Ab dilution/concentration) 100ug total cell lysate/lane protein transferred to PVDF membrane membrane blocked in 6% milk powder in TBS+0.1% tween20 for 7 hours at room temp. Primary antibody diluted in TBS+0.1% tween 20 at 1ug/ml Blot incubated in antibody overnight at 4oC Blot repeatedly washed in large volumes of TBS+0.1% tween20 2nd Ab = 1:3000 [anoter compagny] swine anti-rabbit-HRP Blot repeated washed in large volumes of TBS-T Previous blots detected with standard ECL - no signal therefore the blot I sent you was detected with ECL plus. thanks for your help
Asked on Jul 12 2006
Thank you very much for your reply and detailed protocol which is very similar to the one we recommend. In our experience too much blocking can prevent the antibody from binding adequately to the membrane unfortunately. I would therefore recommend to block the membrane for 1 hour only and as you have done in the past to incubate the primary antibody in TBST only. We also have very good results with 5% BSA in the same conditions. If you still have problems with a reduced blocking time please let me know by e-mail or do not hesitate to call me and I will arrange a refund on your order immediately.
Answered on Jul 13 2006