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primary antibody for western - (Mar/01/2010 )

hi,
Is it possible that an antibody works for immunofluorescence and not for western blot????
in fact, my immunofluorescence was good even the intensity of the protein was not strong! but by western blot, i could'nt see my protein even by repeting several times. and improving all the steps of solubilization and blotting!

-luciana-

I know anecdotally that it's possible -- I was looking into having antibodies raised against custom peptides, and this was one of the caveats -- that the antibodies might not work for all applications. They said, for example, that a particular batch might work fine for ELISA, but not for westerns, or vice versa. So, I think it's possible, though I've never run into this problem personally.

-HomeBrew-

Your antibody might not recognise the reduced protein. Does it say on the data-sheet that is validated for WB? If not, could you try a different clone of the same antibody (I'm assuming monoclonals here), or contact the company for their advice?

I made monoclonals in the past, and had clones that worked beautifully in WB and others didnít although they did work on ELISA and IF.

Hope this helps.

-almost a doctor-

A good point, almost a doctor! The denaturing environment of SDS-PAGE may destroy the epitope your antibody is recognising in immunofluorescence. Perhaps you should try native (non-denaturing) PAGE? As a test, you could try a dot blot of your sample -- if that works while SDS-PAGE/western blot doesn't, a necessary three-dimensional epitope is probably being lost when the protein is denatured.

-HomeBrew-

almost a doctor on Mar 2 2010, 10:30 AM said:

Your antibody might not recognise the reduced protein. Does it say on the data-sheet that is validated for WB? If not, could you try a different clone of the same antibody (I'm assuming monoclonals here), or contact the company for their advice?

I made monoclonals in the past, and had clones that worked beautifully in WB and others didnít although they did work on ELISA and IF.

Hope this helps.

hi,
my antibody is goat polyclonal! in the datasheet, it can be used for WB. with secondary antibody donkey anti-goat HRP.

-luciana-

I've seen a similar issue with a mouse monoclonal against LAMP1. The antibody seemed to work well for IFA, but for a Western, it didn't detect anything. We figured it was because we weren't getting enough protein onto the membrane, because when he lysed 10x more cells, the WB worked with the the same primary and secondary.

Other than lysing new cells, you could try to get a more sensitive HRP substrate. I use SuperSignal West Pico for most of my Westerns, but if I need something more sensitive, I go to the SuperSignal West Femto kit, which seems to be ridiculously sensitive. Another option, if you think it's the signal and not the actual antibody, would be to try a biotinylated secondary antibody, then use an HRP-conjugated avidin or strepatavidin.

-fishdoc-

HomeBrew on Mar 2 2010, 12:54 PM said:

A good point, almost a doctor! The denaturing environment of SDS-PAGE may destroy the epitope your antibody is recognising in immunofluorescence. Perhaps you should try native (non-denaturing) PAGE? As a test, you could try a dot blot of your sample -- if that works while SDS-PAGE/western blot doesn't, a necessary three-dimensional epitope is probably being lost when the protein is denatured.


In fact, this antibody of membrane protein worked for western blot before (laSt year), we could see the protein of interest. so, i don't think its matter of sds denaturant!! we need to load the gel sds to see it!!

-luciana-