I wish to submit a sample to SDS-2D following western blotting. So I´d like to know if is possible to remove that sample used at botting for another experiment, like mass spectrometry.
If the protein is in the gel is possible. But if you transferred to a membrane not.
There are descriptions of MS after WB. I've never done it, though you essentially cut the band, strip the membrane of antibody, digest the protein on membrane, and perform MS. Off course this is a dramatic reduction of the steps required, but it is doable. For example, look at: PMID: 17938404.
As I understand you need to do the Western blotting to localise your protein. I'd run the sample in duplicate on the same gel, then cut the gel and process one of its parts following WB procedure, and the other would be stained with comassie. After that I would cut the area coresspondind to the spot on the blot from the gel and use it for MS.
It is quite common some protein staying in the gel after transfer. If you stain the gel with coomassie you will detect the protein that probably is enough for mass spectrometry
There is a place : Here http://www.proteomefactory.com/knowhow/itproteomics/itproteomics.html . In that place they offer a service, where I understand it is possible send samples transferred to PVDF membranes and they process by mass spectrometry. I need also send samples there. I wrote them last week to ask for the service but I haven´t recieve any answer¡¡.
Juliana,
é possível, sim. uma vez que a proteína ainda esteja no seu gel (se ela não tiver caído com a eletroforese), vc pode fazer a corrida normal de WB e ao invés de transferir para nitrocellulose ou pvdf, corar este gel com comassie para localizar a faixa da banda de interesse e mandar para a espectometria.
se precisar de ajuda com o protocolo, é só me avisar!
boa sorte!
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