Help: How to treat histone for running SDS-PAGE - (Sep/11/2006 )
I usually run invitrogen 4-12% bis-tris gel. Denature proteins by adding DTT, LDS loading buffer and heating. But It looks that I cannot do it for histone.
My histone is from Sigma. It is said that "Normal SDS-PAGE condition give anomalous results"
on the product sheet. And they suggest "An acid-urea-detergent system should be used and the polarity of the poles reversed". I am confused about it.
A nice guy told me "This is because SDS carries a negative charge and SDS tends to form micelle aggregates at the concentration that you normally use for SDS-PAGE. Histones carry positive charges. When you mix these two, they form instantly aggregates due to charge-charge interaction, which can be complicated.
Acid-urea system is used to run histone as positive charged protein. Urea keeps them from forming aggregates. You should reverse the field direction such that histone will migrate towards bottom of the gel. (normally you connect red wire to the bottom, but that is for anionic SDS-protein complexes). "
That makes sense, But I am still not clear about how to do it.
In our lab, we have Invitrogen Xcell sure lock system for running gel. We buy precast Nupage gels from invitrogen instead of making them ourselves. I wonder if anybody has the experience on using Nupage gel to run histone. Or anybody can give me a protocol for it (Donot have to use Invitrogen gels). I think there are lot of guys who are running histones here.Hopefully, I can get help from you. Thanks a lot.
The "anomalous (sp??)" result that Invitrogen is talking about is that
the Histones will not run in the correct order.
That is, their order should be from top to bottom:
H3, H2A, H2B, H4
However, due to charge anomalies the order of H2A and H2B and maybe H4 can
be interchanged H3 is always constant.
It is recommended to run histones on a special gel for proper separation that is composed of
acrylamide:bis at a ratio of 30%:0.15%.
Howver, I simply used a normal 17.25% polyacrylamide gel with common lab polyacrylamide and
got great separation.
The EXACT order of the histones though remains a mystery.....