Help: How to run Histone on SDS-PAGE gel? - (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.
Does anyone know how to treat histone before runing SDS-PAGE?
Thank you so much.

---KL

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).

Thank you so much for your help.

This is a good explanation.
I am still not very clear about how to do.
Sorry for this. I have never run such proteins before.
So what I should do is like this?

1) dissolve histone in acetic acid.
2) add urea
3) add LDS loading buffer (4x)
4) Heat

I should reverse the field direction by switching the red and black cable before running.
Do you have any idea about which running buffer should be used. Usually, I just use MOPS.
Thanks again for your help.

----kL

try this one: Mold DE et al (1983) Anal Biochem 135:44-7. and ref within.

Thanks a lot.

You mean it is no way to run histone on Invitrogen Nupage gels.
I need make them according to the recipes anyway.

check this thread:

http://www.protocol-online.org/forums/inde...showtopic=17321