Determining a protein's antigenic property - (Aug/03/2009 )

Dear all,

I've been in the nucleic acid field for a long time but this is the first time i'm doing protein work.
I have an ORF of unknown function, and would like to know if this ORF produces a protein which is antigenic?
I have successfully cloned the ORF in the correct orientation in pBAD-TOPO. After this I'll be doing pilot expression and SDS page to check the protein size and expression.

My question is:are the next steps correct?

I should do a Western blot (denaturing/native??) what's the difference? So I use SDS-PAGE gels to separate my protein and transfer to a PVDF membrane.
Then I use mouse sera (from our mice which had been infected with my microb) to probe the membrane.
But how would detect positive results?

Since my protein is unknown, there isn't an antibody for it. Is it possible to use an anti-mouse secondary antibody to detect if the mouse sera can interact with my protein?

Thanks for any input, people. Have a nice day...

Chris

chrisbelle on Aug 2 2009, 11:14 PM said:

Just seeing that mouse sera gives a positive result does not mean anything, it could be entirely non-specific result.

1. You can do two identical membranes with your pure protein run on SDS-PAGE, incubate one with anti-mouse sera and incubate another with anti-mouse sera and your pure protein (competition), if you see diminution of signal in the competition expt, you can take that as your protein is antigenic. But this is not surefire way.

2. Raise antibody to your protein. You can inject your purified protein (or a putative antigenic peptide thereof) in a different species (rabbit, sheep etc), and use that sera as specific anti-sera. How to find antigenic peptide? (Here). How to raise antisera? (Here).

Dear all,

Cellcounter, thanks for your input. I'll need to think about that.....
In reference to my 1st post, since the ORF is in a database but the ORF is uncharacterized, is there anything else that I can do (preferably bioinformatically) that can contribute to characterizing this ORF?
Would reporting the bioinformatically-predicted protein size, PI, etc. alone without other experimental procedures constitute as initially characterizing the ORF?

many thanks!!
Chris

What is the source of the orf? If it's bacterial, can you make a deletion mutant? If you can, you can use the mutant as a control for your westerns (band in WT versus no band in mutant). Further, you can use the mutant strain to adsorb out irrelevant antibodies from your whole-bug sera, leaving behind just those that react with your protein.

HomeBrew on Aug 25 2009, 11:06 AM said:

Hi HomeBrew,

Oooo, my ORF is from a yeast. Making a deletion mutant will be quite a lot of work and time. I would like to do deletion of this gene as well, but due to time constraints, I need to "initially characterize" the ORF. I thought testing the ORF to see whether it reacts with sera would constitute testing its antigenicity, until Cellcounter replied me.

So I would like to rush an initial characterization, any characterization that would give me just a little more info about this ORF for a publication.
Therefore, I'm asking if the bioinformatic thingy is possible and considered as some sort of characterization, albeit very initial.

Thanks people!!

Chris