native or denatured protein? - for antibodies (Nov/13/2007 )

What kind or protein do you use to get Antibodies, native, partial denatured or denatured?

I’m going to use the Abs mainly for western blot

thanks in advantage

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Your Western blot will be picking up denatured sequences, so your best bet for antibodies is to immunise with denatured protein. You can use whole protein or you could try a partial digestion and immunise with peptides. If you use whole protein, remember to make sure the protein can't renature: treat with a disulphide blocker like iodoacetamide.

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Hi princess,
I agree with Swanny that it is better to immunize with denatured protein since your objective is to detect by western blot.
But i don't think it is a better idea to digest the protein or immunize the peptides because digested proteins may lose highly immunogenic epitopes and all the peptides may not be good immunogens
all the best
Leelaram

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OK, Thank you very much for your advice.
I will try denatured protein

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we've made antibodies both ways. if you make polyclonals then it doesn't matter as much because some of the species of antibody produced will bind to an exposed epitope when doing western blot or elisa or immunohistochemistry.

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I agree with mdfenko. If you are making polyclonal antibody then it usually does not matter. In that case, I would rather use simple GST-fusion protein as antigen. Right now you just want the Ab for WB, but what if in the future you want to use it for other purposes like IP or IF... and it turns out that you need Ab for undenatured protein?

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