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Immunohistochemistry - In Situ Hybridization: Forums
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Immunoportal.com :: View topic - "Far"-IF staining?
The concept behind Far-Western blotting is using a non-antibody protein to probe for binding partners adsorbed to a membrane. Then use an antibody to detect the first protein.
I'm interested in basically doing the same thing using immunofluorescence. Basically, I have a soluble protein X and I'm interested in which cells manufacture its binding partners Y1, Y2, Y3.... in a particular disease state.
I've tried an affinity chromatography approach before, but my worry is that I'm getting irrelevant interactions and I have no way to quantitate how much of the interaction is going on, as would be possible using an IF approach.
I'd like incubate tissue sections with protein X, then incubate with primary antibody against X, and finally secondary antibody against the primary.
Has anyone tried this before?
Fri Jan 27, 2012 3:17 am
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1839
Post subject: No but....
A long time ago, we cut fresh frozen tissue sections and incubated them with blood cells and it worked:
http://pubget.com/paper/7714332
So your suggestion should work.
Of course, if the protein binds with it's ligand, you may have to briefly fix before antibody incubation, to stabilise the bonding.
Interesting....
Mon Jan 30, 2012 9:09 am
salton
Rank: Member
Joined: Dec 02, 2011
Posts: 3
Post subject: Re: No but....
Carl wrote:
A long time ago, we cut fresh frozen tissue sections and incubated them with blood cells and it worked:
http://pubget.com/paper/7714332
So your suggestion should work.
Of course, if the protein binds with it's ligand, you may have to briefly fix before antibody incubation, to stabilise the bonding.
Interesting....
Are we talking a 10 minute post-fix with 10% NBF or something like that? I should mention that I generally need 10 minutes of pepsin/acid/37C or 20+ minutes of boiling citrate buffer to perform antigen retrieval for this protein, but that's for a 4 hr fix in NBF and dehydration to paraffin. Perhaps a light post-fix would not be too much of an issue...
Tue Jan 31, 2012 2:27 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1839
Post subject: ah!
I was assuming that you would be doing frozen sections.
So, in the 1st instance perhaps try fresh frozen sections: incubate with protein binding partner.
Then briefly fix before addition of primary Ab?
That way, autolysis wll be minimised?
Also, your binding partner, in the section ( hopefully), is in it's native state for the binding interatction?
I would have thought that doing what you suggest on antigen retrieved Pwax sections would not work.
However.......I would like to be proved wrong.
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