Western blot problem - Western blot (Feb/15/2005 )
Hi! I trying to do western blot assay with denatured sample.
It didn't show any band after western blot.
You guys think that 8M urea inhibited antibody reaction in western blot?
I don;t know it... please help me.
i think 8m urea probably would denature your primary antibody and secondary antibody. why did you use it?
Thanks for reply...
Then should i dialyzed sample first, then do western blot?
My protein was insoluble, that's why i used.
What should i do the next?
for a westernblot under denaturing conditions, having your sample in 8M Urea should pose no problem per se, works very fine for me.
I don't think that some µl of 8M Urea/well of PAgel will denature your 1st and 2nd Ab... the urea should be almost completely removed or dilluted to infinity at the point where an Ab "meets" the membrane....
think of all those volumes used (transfer-buffer, then blocking buffer, then 1st Ab.....)
you should check if your samples hold proteins at all by coomassie/silver stain first, and then check your membrane after transfer by ponceau-staining.
and no offense meant: sometimes it shows that the membrane was on the wrong side of the PAgel while western-transfer....
Urea will denature your protein, it might as well change the epitope structure, and inhibit the primary antibody from binding.
Of course you're right, but in denatureing SDS-PAGE this isn't an issue, since all proteins get denaturated and epitopes detected by denatureing SDS-PAGE and western blotting are primary epitopes solely anyway, since secondary and higher structures are dissolved...