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Multiple antibody blotting - western blot - (Feb/09/2009 )

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hello to everybody.
i'm planning to incubate the membrane overnight with 3 different antibodies made in the same specie.
the three protein are different in size .. so I don't understand why it shouldn't work

does someone experience with this ?
thank you Gianluca


I would be careful with that.
Sometime some antibodies also recognize an other band, it's considered as normal and we don't care (unless if you incubate also with an other antibody that should recognize something close to the non specific band.)
Sometime one of your protein of interest make aggregates, dimers, then you would get two bands with only one antibody, which could also be a problem if you incubate several antibodies. You won't be able to say if the band you see is one of your protein of interest, or one non specific band of an other protein of interest, or some dimers or aggregates of the third protein of interest.
I would do a simultaneous incubation only if I would already have done single incubation and confirmed that I only get one band each time.

-little mouse-

This could get ugly, fast. If all the sizes are different, why not cut your membrane? I run marker on both sides of the gel, ponceau after transfer and cut the membrane so I can incubate all three of the correct sizes with the desired antibody at the same time but not by mixing the antibodies. Just be sure to block after cutting the membrane so antibody doesn't bind the edge where you cut. I know western blotting is time consuming and everyone wants the answer to their experiment now (ok, only if it's the right answer) but this technique is one that will generally pay off the best with patience. Don't push too fast or with too much antibody (especially mixing them!!) or you'll wind up with a black piece of film.


sequential use of antibodies should be done. Results obtained with antibody cocktails should be invalid.


Hi there, Question to Rkay, you say you transfer, do stain on the blot, then block and then cut. How do you cut the blot (vertically, horizontally?). What do you use to cut the blot?


cutting the membrane can be done vertically and/or horizontally depending upon your loading of samples on the gel. first wrap the PVDF membrane in saran wrap then draw a line with a marker pen on the site of cutting then cut by a sissor. I always do this to detect multiple proteins on the same blot with good signal without the need for antibody coctail, or stripping. In addition it saves time and consumes less amout of antibody (save mony as well)
Attached File


I do it all the time, provided the antibodies(monoclonal) are really really good. i dont think why people should be worried of this technique its really helpful when you are running out of precious patient samples.
i have used max 4 antibodies in a single membrane once, giving me only 4 specific bands.
this works with mouse antibodies from BD biosciences, but did not work with rabbit antibodies from cell signaling. would not recommend this approach with goat antibodies from santa-cruz :lol:


rajgene on Jul 1 2009, 03:03 PM said:

i dont think why people should be worried of this technique

you answered your own question when you listed a random bunch of antibodies not to do it with - its an invalid technique as it is based on dumb luck - antibody/antibody interactions are seriously unpredicatable and best avoided



interactions of antibodies is not seen only in cocktails, but also after an incomplete stripping of one antibody followed by reprobing with another in WB where a potentially misleading bands can be found on the membrane


yes of course it has to be standardized for each needs. but i am 100% sure it always works provided you know what your doing. its no dumb luck..all you have to do is just give it a try and save valuable time and money. :)

P.S in my previous post i just hinted my experience with different antibody sources based on the quality of antibodies provided by the manufacturer.(BD and cell signaling being the best)

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