Stripping of nitrocellulose membraned - (Jan/29/2006 )

Hi all,
I want to reprobe a nitrocellulose for a phospho-specific antibody. What is the most gentle stripping protocol that will ensure that most of the phosphates will remain bound to the membrane?
Thank You!

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Hello,

345 uL of beta mercapto ethanol
10 mL SDS 10%
complete to 50 mL with tris HCl 63 mM pH 6.7

30 minutes at 50°C
under gentle agitation.
wash extensively, and block again.

you should still get a signal, however a little weaker.

next time : IB first with anti phospho specific, then strip and blot again with your "anti-complete" .

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what is meaningof last sentence

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Ok I agree, I was hurry up.

OK you know that when you want to show that there is an increase or decrease of the phosphorylation of a protein, you have to detect also te protein independently of the phosphorylation state, to show that the expression level is not changed.
then you have to show phosphorylation, to strip and to show protein expression.


As stripping is removing antibodies bound on the surface of the membrane, but also some proteins transfered on the membrane you should better start with the lower signal (phosphorylation) to get more chance to see it. If you see a phosphorylation, you should then be able to see protein expression level even after stripping, because the signal is stronger

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Just a question, whatt are your transfer set up for a nitrocellulose membrane. For the PVDF I do the transfer 1,5 hour at 250mA.

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for a protein at 30 kDa : 08 mA/cm2. 1 hour

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have you guys try to label the protein with antibody conjugated with fluorescent? if you use different colour fluorescent, you will get these bands under different filter. however, you needs a equipment to read the results.
the good thing is you do not need to strip your blot and lost some bands on the blot.

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Yes, but I think it's not helping the antibody if there is already other antibodies bound on the antigen...
and one could imagine that more antibody n°2 will bind where no antibody n°1 didn't bind, it means the binding of antibody n°2 will no more corelate with the expression level of the protein, but maybe also with the non phosphorylation state

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