cell fractionation and obtaining pure nuclei - (Jan/14/2008 )
I am doing cell fractionation on Hepg2 cells. As a detergent i use TKMS with 1% NP-40. The detergent was good for extracting the membrane fraction protein but the yield from nuclear fraction was not so good. I've heard that NP-40 is not a good detergent for the nuclear protein because it's not able to break up the nuclei. Is that true and if so, can you suggest a better detergent in this case? Also, how much cells do you need to obtain a decent (~300 ug of nuclear protein for IP)?
i got very efficient preparations using a protocol derived from dignam et al. (monitored by western blot and efficient on in vitro transcription assays
You can find it here
-total cellular extract
-proteins from cytoplasm and proteins from nucleus in separate tubes
to get more activity, i do a wash with buffer A without NP40 to the first pellet.
It means :
resuspend cell in 2 volumes of buffer A +DTT+PMSF
spin 2500g 15' 4°
resuspend gently the pellet in 1ml buffer A + DTT + PMSF
don't add NP40
spin 2500g 15' 4°C
then go for standard protocol.
it enhances transcription activity, probably by finishing removal of cytoplasmic inhibitors.
so NP40 at 0.05 to 0.1% is not strong enough to "dissolve" nuclear membrane, but trust me, combination of buffer B and C is enough to break cell nuclei.