reusing PVDF membrane for different antibodies without stripping - (Nov/23/2007 )
Hi,
I would like to ask (you) for some info about stripping a PVDF membrane.
1. Do you have any experiences with (several times) re-using PVDF membrane which was incubated with an antibody again with an other antibody without any stripping?
For example, if I would have four different antibodies to four different proteins, could I incubate PVDF with one antibody, then wash the PVDF (with TBS-T or NET buffer), then with the second antibody, then wash. . . etc. or there can be a problem of non specific blocking some proteins with secondary antibodies or anything else?
2. Antibodies bind to protein (on PVDF) by weak interactions (not any covalent bound), so why is it actually necessary to use quite harsh condition (stripping in SDS, mercapto-ethanol, high temperature) to remove antibodies from membrane (from proteins on membrane)?
Many times, I needed to do stripping even more than one hour to remove antibodies from PVDF (at 60dgrC). So I would like to know, whether someone has experience with stripping at room temperature for short time with classical stripping buffers or some special buffer.
I am afraid of whatever stripping PVDF membrane because proteins of my interest have very low concentration and are hardly detectable with any antibody.
Thanks for your advices.
vic
I would like to ask (you) for some info about stripping a PVDF membrane.
1. Do you have any experiences with (several times) re-using PVDF membrane which was incubated with an antibody again with an other antibody without any stripping?
For example, if I would have four different antibodies to four different proteins, could I incubate PVDF with one antibody, then wash the PVDF (with TBS-T or NET buffer), then with the second antibody, then wash. . . etc. or there can be a problem of non specific blocking some proteins with secondary antibodies or anything else?
2. Antibodies bind to protein (on PVDF) by weak interactions (not any covalent bound), so why is it actually necessary to use quite harsh condition (stripping in SDS, mercapto-ethanol, high temperature) to remove antibodies from membrane (from proteins on membrane)?
Many times, I needed to do stripping even more than one hour to remove antibodies from PVDF (at 60dgrC). So I would like to know, whether someone has experience with stripping at room temperature for short time with classical stripping buffers or some special buffer.
I am afraid of whatever stripping PVDF membrane because proteins of my interest have very low concentration and are hardly detectable with any antibody.
Thanks for your advices.
vic
we do re-use w/o stripping if IgG of different species are used, f.i. 1st immunodetection with rabbit primary Ab then anti-rabbit Ab, then deactivation of HRP with 3% H202, 2nd immunodetection with mouse primary Ab and detection with anti-mouse IgG; could be continued with donkey, goat or other species
We also do it, but we quench HRP by azide (toxic). But make sure that the subsequent antibodies have to be from other species than the ones used previously. For example: first probe for protein X you used mouse anti-X and goat-anti-mouse. Then the second probe for protein Y you cannot use either mouse-anti-Y or goat-anti-Y as primary antibody. You have to use, for example, rabbit-anti-Y and goat-anti-rabbit. Thrid probe will be even more restricted and so on and so forth. Also, after quenching, I don't know with H202, but we have to wash the membrane profusely to get rid of the azide, otherwise it will interfere with the subsequent probes.
As for stripping, we no longer use the SDS-2ME-high temp protocol. Now we buy PIERCE stripping buffer. Can be use at room temp, 30-60min. And no 2ME smell, either. Very nice.
hi,
i do agree with our forum members, but the draw back is both sodium azide and H2O2 mediated inhibition is reversible. upon withdrawal of the inhibitor u can see the HRP function as background.
follow the attached paper
sravan payeli
I always strip my PVDF membranes at room temperature:
- 10 min in water
- 30 min in NaOH 4N
- 3x2 min in water
- saturating step (at least 1h TBS-Tween 0,5%, milk powder 3%
- usual washing steps and antibodies incubation
Hope it will help you... 
As long as the size of the proteins are sufficiently different, cut your membrane after transfer and ponceau staining. I do IPs where I need to look for four different proteins (the IP itself ~75kDa, a positive control ~30kDa and two interacting ((hopefully)) proteins ~50 and 110kDa). I just run marker on both sides of the gel so I can put down a ruler and make a perfectly straight cut from one marker to the other. This way I can probe one experiment/membrane for all four without having to worry about the signal from other antibodies.
have you tried to mix your 1st antibodies for these four different proteins together if they require the same 2nd antibody?
I once mixed 5 1st antibodies (the MWs of target proteins are well seperated) and it worked pretty well.