Blotting - (Dec/17/2012 )

Hi all,

I have done western bloting with my recombinant protein
it gave good reaction against hyperimmune serum raised in rabbit
but when I used the antigen to screen it against infected human and cattle serum I could see no bands

so I went for dot blot with the protein against human and cattle serum at different dilutions at dil of 20, 40 , 80 , 160, 320, there was no reactions , but a faint dot was seen at 640, 1280 diltutions of serum........


what could be the reason for this kind of test result ....?
so if iam increasing my dilution to 1280 in western blot . would I get result.....?

With your dot blot assay you would expect to see a titration of the intensity of the band with the lowest dilution (1:20, is presumably what you mean) giving the strongest signal, and the signal getting progressively weaker from there.

The antigen you have - is it a sequence that you would expect to find in a native state or would it only be recognized in a denatured state?

Actully there was no signal at 1:20 but i could see it only in 1:1280 dilution of serum

earlier researchers have shown that the recombinant protein is recogonized in refolded form.... as n my case too but iam not getting reaction

Gunalan on Tue Dec 18 05:24:22 2012 said:

The dilution thing shouldn't happen like that, I would be very worried that there is something wrong with the assay for that to happen. Perhaps there is contamination on your blot or is this reproducible?

Refolded=native... you won't detect it on a denaturing blot in that case.