The best way to get nuclear proteins from mammalian cells - How to break your cells? (Feb/17/2005 )

Can anyone tell me, what´s the best way to extract nuclear proteins from mammalian cells for SDS-PAGE? And how much cells do you use per well?

I have tried the following method: tryptinize the cells, suspend into medium, centrifugate 1500 rpm for 5 min, sonicate the pellet in loading buffer (containing ME)..and then straight to the gel..

I have also tried to harvest the cells..

It seems though that the stuff is stucked into wells and doesn´t move in gel very well. The protein I´m trying to seek is still only 60kDa...So I think the major problem is to do the right kind of sample.

Should I use protease inhibitors like PMSF or CLAP? Or am I using too much cells (50 000-100 000 cells/well)? And in which method the cells will break but your protein won´t?

If anyone has good advice I´d really appreciate it!

Thanks!

Hi! If you are only interested in nuclear proteins, is better to do nuclear extracts. In the protocols page here there are several protocols. The one I use is quite similar to the one which is entitled "Quick and dirty". If you want to obtain better quality lysates you should use the Digham method (the third one in the listing) which is a little more laborious.
The advantage of preparing nuclear extracts is that your protein will be more concentrated in them that in whole cell lyssates (assuming that your protein is strickly nuclear).
I normally follow the schreiber method for nuclear extracts, using 1 or 2 100 mm dishes of confluent cells. I measure the protein by Bradford assay and load 10-50 ug in a gel, depending on how abundant my protein is.

Hope this helps!