Hello Visha,

did you blot the whole blotting paper. membrane proteins tend to aggregate during the stack phaase. perhaps you could blot the whole paper to check if there were aggregated, If so, i will suggest heating your samples at 37 degrees for 15-30mins instead of the usual 95 degrees for 5-10mins.

There is a previous thread expanding on membrane proteins in Western Blot, which indeed is tricky. Here is the link:

https://www.researchgate.net/post/Membrane_Protein_Western_Blot

A lot of interesting suggestions can be found there, along with some links to relevant publications. Good luck!

Hi, 

I was wondering whether you solved the problem. We see a lot of "background" with the anti-Flag M2 antibody in immunofluorescence although we know that the flag tag is not expressed (Western negative; control cells show similar staining pattern). Is there a lot of background and unspecific staining when using anti-Flag tag antibodies (specifically M2) and how can it be overcome (fixation of cells, antibody dilution?)? I find a lot of evidence that people use successfully M2 for Western blotting; how about cytometry and IF?

Hi Thomas,

Yes, I tried heating protein lysate at 37°C for 30 min which solved my problem.

 I have also used the M2 antibody for Immunofluorescence and it did not gave any background (I have also used same antibody for FACS and western blot).

I have used 4% Paraformaldehyde for Fixation and Triton X100 (0.1%) for permeabilization 10 min each at 4°C and blocking in clean 5% BSA overnight for IF and 30 min for FACS.

Which fixation protocol did you followed for Immunofluorescence and FACS?