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Cell fractionation and isolation of nuclei


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#1 fred197237

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Posted 04 June 2009 - 09:16 AM

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?

#2 dan_

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Posted 22 June 2009 - 11:14 AM

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.



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?






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