denature a protein of interest - (Jul/20/2005 )

Hi,

my antibody can't bind with my protein of interest during IP process, but the antibody can perfectly recognize it on western membrane. No matter how I change the binding condition the same thing keeps happening.

So now, i suspect that the native structure of protein is not accessible to antibody. Probably I need a condition in which protein of interest is in a denatured form but antibody is functional.

My plan is: I could denature the protein first by SDS, then reduce SDS concentration by dialysis, then add antibody. I guess I could reduce SDS concentration to 0.1%, which is still ok for antibody.

Think about SDS-PAGE, we denature protein, after which the proteins are in an environment that contains 0.1% SDS.

Will it work? I'm afraid that in solution, the protein will fold back when SDS concentration is reduced......

I need some advice from you
Thanks a lot!

I wouldn't go with SDS, as it's very difficult to dialyse away from protein. Once it binds, it's unlikely to leave. Instead, I'd try guanidinium. Concentrate your protein as high as you can get without aggregates, then denature with 6M Guanidine-HCl. You then dilute that into a solution containing your antibody. If you're set on SDS, you could do the same thing with dilution and not worry about refolding (antibody would have diffusion-limited access to unfolded protein until it either refolds or aggregates). Antibodies may even be stable in 6M Gu-HCl (anybody know?)

The downside with either method is that you may aggregate your protein of interest when you remove the denaturing agent. If you have access to a fluorometer, you can check for aggregates by measuring light scatter, if it's important.

Out of curiosity, what's the purpose of the IP? Are you trying to pulldown another interacting molecule, or is it a purification step? I ask because a denatured protein is not going to behave in the same way as the native form, so any interaction or activity beyond antibody binding could be lost.

Also, whether the protein refolds in solution is another matter entirely. It may be able to do so on its own, or it may require chaperones for proper folding.

One easy thing to try would be to boil the protein then cool quickly on ice. Then there is no need for dialysis or other chemicals which can interfere with antibody binding.

Thanks a lot to aludlam and ajames:)

Maybe I should post this topic on chromatin board
Actually I'm dealing with chromatin immunoprecipitation, and I got problem in protein purification step. In this case, I don't care about the protein interacting partners or its native structure, I just want to purify the protein without releasing the DNA fragment bound on it.

As the protein-DNA crosslink will be reversed by heat, therefore I can't boil the sample to denature protein.

I plan to incubate my protein sample in a denaturing environment, then dilute it so that when antibody is added in, it won't die.

SDS? Gu-HCl? which one is better?

For your purposes, I'm guessing neither is optimal. SDS will coat the protein with a net negative charge, which could repel the DNA. GuHCl is a safer bet, but I don't know whether it affects the ionic state of the protein, and the tertiary structure may be important for binding. Only way to find out is to try and see.

If you try guanidine, you could also try 8M Urea, which has similar effects but may be a bit gentler than Gu-HCl. Also, you may not need to go to the upper range of either of these reagents. I've gotten efficient denaturation of a model protein with 3M Gu-HCl, and some proteins are purified in the presence of 2M Urea.