I want to perform a western blot of a native page. I tried with normal tris transfer buffer with methanol and it did not work. Since it is a native page, i wonder there is no SDS to charge the proteins. So i want to know whether there is any modification in transfer buffer for western blotting of Native PAGE ???
Normally when you run a native gel, you want to do an in-gel enzyme activity assay, not blot it. In principle, if you run a native gel, proteins with a negative charge will migrate into the gel, proteins with a positive charge will not. Since we usually transfer blots also from negative to positive electrode, in principle that should work, but the charge will be very small compared to proteins where hydrophobic amino acids are decorated with SDS. You may need to blot a lot longer.
Also, remember that there is only one type of denaturing gel with SDS (except for the presence or absence of reducing agents in the sample buffer), but there are many different native gels. They can be acidic, neutral or basic...depending on your protein of interest. You have to know a lot about your protein before you try a native gel, and like I said before, usually it is not with the aim to do a blot afterwards.
It would help if you explain whay you try to achieve.
Thanks Jurgen.
Well I am working on a homotrimer receptor protein of 150KDa. With SDS we see some faint band of monomer size in western blot. In native gel we do obtain bands at 150KDa, to confirm the same I thought to go for a WBlot with specific antibody.
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