Cell fractionation and isolation of nuclei - (Jun/04/2009 )
I'm isolating the nuclei of HepG2 cells using homogenization with the douncer, spinning at 1000 g obtaining crude nuclear fractions and then loading the CNF on sucrose gradient. BUT when i check with ER marker calnexin i find the nuclear fraction has even stronger bands than membrane fractions. That means that ER is not separated from nuclei. Any suggestions on how to obtain a nuclear fraction pure of ER?
-fred197237-
Hi,
Qiagen has a kit that work well for this application : Qproteome cell compartment kit.
I presently use this kit I have always clean nuclear extract.
Hope this can help you.
fred197237 on Jun 4 2009, 09:16 AM said:
I'm isolating the nuclei of HepG2 cells using homogenization with the douncer, spinning at 1000 g obtaining crude nuclear fractions and then loading the CNF on sucrose gradient. BUT when i check with ER marker calnexin i find the nuclear fraction has even stronger bands than membrane fractions. That means that ER is not separated from nuclei. Any suggestions on how to obtain a nuclear fraction pure of ER?
-dan_-