Hi, dear everyone. I had a problem with the nuclear extracts preparation. I used the following protocol.
Cells (107) were washed twice with cold phosphate-buffered saline and the cell pellet was suspended in 400 μL hypotonic buffer A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol [DTT], 0.2% Nonidet P-40 and 1X protease inhibitor cocktails) for 10 minutes at 4°C.
Nuclei were prepared by microcentrifugation for 5 minutes at 4°C.
The nuclear pellet was suspended in 75 μL buffer C (20 mmol/L HEPES, pH 7.9, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 1X protease inhibitor cocktails) and incubated for 30 minutes at 4°C with brief mixing.
The mixture was microcentrifuged (15 000 cpm) for 15 minutes at 4°C.
The protein concentration was measured using the Bradford assay.
The question is different cell lines giving different results after buffer C is added. I got very sticky pellet which couldnot be resuspended for some cell lines. for others, the pellet was easily resuspended. This is the same as last time when I was using the kit from Active Motif. I failed to get nuclear extracts that time.
According to successful experience from my labmates, sticky pellet might lead to good result. I want to know what is in that pellet, genome DNA? how to explain the phenomenon which happened in my experiment.
Thank you very much. I appreciate every reply.
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