Stripping problem - (Sep/18/2014 )
Iam using thermo scientific stripping buffer to strip the membrane for 45 -60 min.The first antibody is anti rabbit. But when I reprobe with the second antibody which is also antirabbit, the second antibody is seen in the same lane as I had the first primary. Since the proteins are of nearly the same molecular weight 57 and 60 kda, Im confused if the results are correct. I have stripped three membranes and Iam seeing the same result. Expertise advice is needed.
I wouldnt try in the same membrane if the MW difference is this little or two proteins that could not be separated, because it is not certain that the signal you seeing is only from the second antibody. May be after stripping you can do ECL and check whether there is any HRP from the first ab, but again there is still a possibility that you might have stripped just the secondary and the left over primary would interfere.
Iam trying to do the stripping at 37deg for 45 min. Will this work.
May be you can run a second gel and running it extensively could separate the bands a little and then you could distinguish the two proteins. sometimes with the shape of the band you can tell whether the signal is from the first probed or the second (this not always works just giving you another possible way to distinguish proteins that resolve equally).
If no option you can do as you said and to be sure of complete stripping the first Ab, may be you can do the secondary alone after stripping and perform ECL if no signal you are safe and proceed with the second probing.
To say this first: I really don't like that Thermo Stripping Buffer. It never satisfactorily removed all the antibodies, as verified with ECL, no matter which recommended protocol I tried. As GNANA pointed out, to be really sure, you could verify removal of primaries by incubation with only secondaries and then ECL.
The hands-down best stripping buffer protocol/recipe I have ever used is incubation in 100 mM NaOH, 2 % SDS and 0.5 % DTT for 1 h at 55 °C. Sounds kinda harsh but it actually works several times on the same PVDF membrane. Have not tried with NC.
As for identification of the bands I can recommend a little trick, in case you are detecting digitally. After you have detected your chemiluminescent signal, you take a regular photo of your membrane at the exact same position. If you overlay those two images in a capable software (like Photoshop) you will have the exact location of your signal on the membrane. And if you do this with the same membrane but different antibodies, you may exactly overlay those two overlays by the dimensions/outlines of your membrane, and see the location of both signals. Hope that's not too confusing.