Nuclear Extract problem - (Jul/01/2005 )
Hi, I am trying to prepare some nuclear extract from mammalian cells, both tissue and cell lines. I have tried several times and used western to try to detect my protein of interest, but obviously, I did not get anything at all.
I am using the pretty standard protocol, hypotonic buffer, Dounce homogenizer, low salt/high salt approach. The control sample, which is total cell lysate using SDS sampling buffer worked well in the westerns, just that I did not find anything at all, the cytosolic fraction, nuclear fraction and even the debris! Anyone has experienced the similar problem before, I need help urgently!
I don't have much experience with nuclear protein extraction, since I've just started a couple months ago with the technique. I've been doing it with tissue (30 mg approximately for sample) and with cells (4-5 million cells approximately) too, and everything's right according to my protein quantifications and WB results. Are you using similar quantities of tissue/cells? How's your nuclear protein concentration? How much protein are you loading for SDS-PAGE? I usually load 30 ug/well. Since you don`t obtain proteins even from the cytosolic fraction, I'd check up the antiproteases in your homogenization buffer.
Well, I did add adequte amount of PMSF plus protease inhibitors in the extraction buffers. My protein of my interest does not show up at all, both the NE of tissue and cell culture. It somehow just disappears. Since I did not see anything on the western, I just loaded as much as I could in the well. Well, I'll have to double check everything then. I have also read I a previous thread about chromosomal DNA containmation that makes protein extraction difficult from the nuclear pellet.
Did you do a positive control for your Western? No, you didn't right?
ALWAYS, incluse a positive control when doing something for the first time.
Most likely, its not the nuclear extract thats the problem but your Western blotting procedure.
Even if a protein is nuclear you could still detect it (if you load alot more) in whole cell extracts.
If you are certain you have this protein in the cells you are using use RIPA buffer to collect protein and load a tone of it (100 micrograms or more) onto a gel. You should be able to detect it.