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[lab2012]

Hello, I am now making nuclear extract for the superose 6 column gel filtration (size fractionation) from HCT116 cells. The filtration needs to load nuclear extract less than 0.5ml but more than 10mg(concentration should be more than 20mg/ml). I have used the following protocol to make the nuclear extract. Tried different nuclei lysis volumn like 50ul, 25ul, 10ul. I adjusted the sodium concentration also. None of them give any significant improvement. My outcome is always around 2-4mg/ml. Is there any way to solve this problem?

Thank you very much!!!!

Cytoplasmic and nuclear cell extracts were prepared from a confluent 10cm plate of HCT116 cells in 400ul hypotonic buffer (10 mM Tris, pH 7.4/1.5 mM MgCl₂/10 mMKCl/0.5 mM DTT/protease inhibitor cocktail tablet) on ice. After 10 min NP-40 (0.1% final concentration) was added, votex and nuclei were pelleted by 10-sec centrifugation at 5,500 × g. The cytoplasmic fraction was transferred to a new tube, and nuclei were washed three times with hypotonic buffer. Subsequently, the nuclei were lysed in lysis buffer 50ul (10 mM Tris, pH 7.4/150 mM NaCl/1% NP40/0.5% sodium deoxycholate/1 mM EDTA/1 mM DTT/protease inhibitor cocktail tablet), votex, then incubate on ice for about 15min, then 14000rpm for 5min. Transfer SN into a new tube as nuclear extract.