Can I test protein denaturation after sonication - (Aug/31/2012 )
I was wondering if anyone has a suggestion to how I can test if my protein of interest becomes denatured by my sonicationprocedure.
My intend is to lyse Hela cells in a mild buffer by sonication to get whole cell extracts for immunoprecipitation studies. My current challenge is that I dont get much of the protein of interest precipitated. Im currently testing different factors, like antibody and so on. However, I would also like to investigate whether the sonication results in denaturation of the protein, but I cant figure out how to test that
Here is what I do:
I sonicate freshly harvested Hela cells in a mild buffer 5x4sec on ice (app30W). No significant foaming was observed, while viscosity and clarity of the sample changed. Next, I centrifuge the extract at 26000xg for 30 min. The supernatant is then used for further studie. I get nice bands of the protein of interest from the lysate compared to pellet, indicating that most of it is released from DNA and cell debris by the procedure. But can I in some way test if this released protein is denatured to some extent??
Thank you for taking time to read my message
To answer your question, well it depends what your protein of interest is and it also depends on the antibody you are using. if you are new to western blot then I must tell you that certain antibodies might bind to different locations of a protein. Also, some antibodies are recommended for western blot but others are recommended for Immunofluorescent or IHC. So it is difficult for us to answer you. I must also tell you that I've been working with cells (specially HeLa) for almost 9 years now and I have never lysed my cells with your method. I know some people don't like detergents but I have to tell you that I have never heard anyone of my colleagues complain about digitonin or CHAPS lysis buffers. and 26000g for 30min??? That is a lot!!! do you use a refrigerated centrifuge? Because if you don't your protein might really get degraded! depends on the protein though. Do you use house keeping genes as control? like Actin or GAPDH? you must use and compare the intensity in your sample and control. You must also normalize your samples to load same amount of protein on the gel.
If you have a relatively pure preparation of your protein of interest....spike it into the sonication buffer + cells before sonication and then sonicate and spin, and spike it into the the mixture at the same concentration after sonication and spin and see if your before and after in the supt. give the same immunoprecipitation results.
If your sonicated spikes have less intensity than your non-sonicated spikes, then the procedure is degrading the protein.
You will have to do a few spike concentrations to hit the right level, so that you can differentiate spike from endogenous protein.