Heavy background on pvdf membranes blocked with 5% skimmed milk and probed with affinity purified rabbit pAbs
I am probing plant MAPK species using commercially available antibodies from Sigma. Following transfer, I block membranes (PVDF) with 5% milk and probe O/N with primary antibody diluted in 1% BSA (Sigma protocol suggest dil. 1:3000 and I use 1:5000). After extensive washing I probe with secondary HRP conj. anti-rabbit (again from Sigma. Suggested dil. 1:5000, I use 1:10000). I have also tried to block before 2ndary antibody as well, but even in this case I get to see multiple bands. So bottom line, I would like to ask if anyone knows about a reliable blocking method for PVDF membranes but also for a good stripping solution.
Thank you all,
George Komis
PS. I am using homemade chemiluminescence, enhanced by p-coumaric acid
Please standardize the concentration of secondary antibody and also please check the concentration of primary antibody suggested by the company. Maybe you need to standardize the concentration of antibody. While washing use PBST for washing and do the washing extensively. Your blocking is fine, side by you can try 10% skimmed for blocking overnight and you can make the dilution of your antibodies in 1% skimmed milk.
good luck
for diluting primary antibody try using 1.5% milk in high tween PBS. This works great for me. For secondary antibody use 2.5% milk in tween PBS. For developing use Thermo Supersignal West femto kit. For washes after primary and secondary antibody give three washes with tween pbs at 5 mins each. Also note to wash the membrane after final wash before putting the development substrate with distilled water(quick washes) to remove any salts. This will help you prevent background.
Cheers!!
We routinely do all incubations (blocking, primary antibody, washes, secondary antibody) in TBS, 5% milk, 0.2% Tween-20. For high background with some primary antibodies, we wash the primary antibody with the same solution but we add 1M NaCl.
Maybe you wash the membrance more times and longer. Otherwise, you add more Tween 20 to washing buffer if you are using Tween.
Hi;
You need to incubate (probing) the primary and secondary antibodies in the same blocking solution (5% milk+ 0.1% Tween). You would better to optimize the Ab dilution factor in each step.
(0.1% Tween TBS 5% Marvel) for blocking step then TBS 0.5% Tween for the first wash then TBS 0.1% Tween should help. But mostly you need to optimize as suggested above also least with a commercial ECL you can get consistant data I've never had much luck with home made and its not that expensive especially when antibodies are so expensive.
I have added marvel for antibody incubations but only for some really dodgey antibodies and that worked OK.
Dear people,
Thanks a lot for your advices. I tried to follow as many as possible, since I had 6 fresh and 6 stripped membranes (following protocol from Lambros). As suggested by Ana, Thi and Melanie, I found that Tween conc. is critical (I got optimal results with 0.5% and there was kind of a dose response comparing "noise" to increasing Tween concs, keeping milk constant). Also, I wish to thank Ana Zen and Steven Russel for their suggestion of using commercial ECL. It surely worked better. Sometimes, it is good to be a little cheap but never save money for a good result. Lambros, thanks for the stripping recipe. I followed it and I got the cleanest results I could dream about. However, there was a loss of signal intensity. Do you think that SDS may also remove substantial amounts of bound protein?
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