SNP effect on protein binding affinity - (Jan/16/2014 )

Dear bioforum friends

I want to know the effect of a known SNP on binding affinity of my protein to another protein. The SNP falls exactly in the binding domain and I think it reduces the binding compared to the wild type protein. CO-IP is not really showing a big difference, I need to have a more sensitive technique. 

BTW, I have cell sample from patients with the SNP and healthy control, I have extracted the protein Cell lysate for my test.

Do you know a better way than CO-IP?

Thank you guys

Niaz

Co-IP is the standard practice for the experiment you described.

Not a direct answer but this may help solidify your conclusions down the line:

thanks for the links, I will check them...

My protein is a trans-membrane receptor and the other protein is it's ligand. You know the SNP is a single nucletide change so the effect is not as huge to be detected in western blot junk bands! but even if there is a small change it proves my hypothesis.

Right... It would be hard to determine a small change due to the semiquantitative nature of western blotting. I'm trying to think whether a dot blot would be good for this. You wouldn't be able to detect the band obviously, but a titration of your Co-IP samples would give you a better understanding whether you have a gain or loss in signal. Probably not the best approach, but I can't think of anything else.

You could always clone your protein with a tag, generate the SNP and purify the protein. You could then purify the protein in a column. Knowing the exact amount of each protein that you begin with would give you a more quantitative approach, but that is a lot of work for a relatively simply question. 

I'm assuming this SNP isn't a known variant? You have tried all the SNP databases to see if anything pops up?

Well, I thought about cloning but it is a long process for an small question as you mentioned, I am wondering how there is not a simpler more sensitive way available, BRET could be another choice but it is going to be expensive I guess....

the SNP is a known variant, but how this can help me to study it's effect on binding to my protein?

Thanks for supports

I was just curious to see if anyone else had established the SNPs function. Good luck!