Confirming antibody specificity

[MicroScope]

Hello,

I have had a synthetic antibody generated for me that is supposed to bind to a specific protein of interest expressed in the pancreas. I have done a Western blot using rabbit sera to blot a membrane containing islet cell extracts.

I chose rabbits that did not have any noise in the area of my protein of interest and am having them injected with my synthetic antibody. When I collect my synthetic antibody from these rabbits I want to confirm the antibody is binding to my protein of interest so I will do a Western and look for a band in the area of my protein of interest (this band should not have been present in the first Western).

I don't think this will directly confirm that my antibody is binding to my protein of interest.
QUESTION: HOW WOULD I DIRECTLY CONFIRM MY ANTIBODY IS BINDING TO MY PROTEIN OF INTEREST?

Thanks very much!!!

[danfive]

You can go to mass spec. You can either punch out the positive western blot signal band and trypsinize and ID by mass spec. Or Immunopurify using the antibody. Then elute the antibody-antigen complex and trypsinize and mass spec.

[MicroScope]

Wow thanks for the quick and very helpful reply!!!


So I would have to run two gels:
1 would be used for blotting with antibody
2 would be used for punching out my positive western band

Is this correct or am I misinterpreting something?

Thanks very much again!!

[danfive]

Originally Posted by MicroScope

Wow thanks for the quick and very helpful reply!!!


So I would have to run two gels:
1 would be used for blotting with antibody
2 would be used for punching out my positive western band

Is this correct or am I misinterpreting something?

Thanks very much again!!

You may do it exactly as you state above. And consider punching out the actual band on the blot paper from the Western blot and digesting and analyzing in mass spec along with the other protein band from the second gel. This way you will know if the digest/clean up procedure can be simplified to just analyzing the protein on blot paper (after your Western blot).

You should also know that if you have a solid band on your gel you can simply use a pipet tip to punch out enough protein for trypsin and mass spec. You can save the rest of the gel band in an epi tube at -80C. That way if you need to trouble shoot the digesting, reducing, alkylating protocol you will have plenty of protein sample as backup.

[MicroScope]

Thanks for your help! It was very useful and informative