Antibody from short peptides - Time saving, but do they work for both West and ELISAs (Nov/20/2006 )

Hi there-

Question for you. I have trans protein expressed in plants. I want to characterize this protein with a western blot and possibly as ELISA.

I have heard of using short peptides for (12- 16 AA) raising Ab. This would save me the time of going the fusion protein route.

Would antibodies made in this manner be able to detect denatured protein on a Western as well as native protein for an ELISA?

Thanks,

-holyrail-

In my experience, yes they are able to detect denatured protein on westerns and the immunogen for an ELISA, but I have not tried native protein.

-WAstate-

QUOTE (WAstate @ Nov 20 2006, 01:47 PM)

In my experience, yes they are able to detect denatured protein on westerns and the immunogen for an ELISA, but I have not tried native protein.

Thanks for the reply.

Regarding your ELISAs - So you're saying that you used a denaturing protocol to isolate crude protein extract that then worked for your ELISA - but your not sure if it would have worked if you used a native protocol.

Thanks,

-holyrail-

I used the short peptide for my ELISA, and that works just fine. I have not tested the antibody against non-denatured full length protein.

-WAstate-

if you use a peptide for immunization, you should be able to use the antibody to detect denatured proteins and the peptide.
About ELISA, of course you will detect the peptide, but not for sure the native proteins. Depend on the refolding of the protein.
If you know the 3D structure of the protein, you can choose smartly the peptide so it is not hidden, it's well present on the surface, and then you select the clone expressing the monoclonal antibody that recognizes both the denatured protein and the native one.

-Missele-