What is the white cloudy precipitation after lysing HEK-293T cells?
My lysis buffer is composed of 1%NP-40, 150mMNaCl, 50mMTris, 5mMEDTA.
After lysing 30min and centrifuge at 1,2000 *g with 30min, the lysate still has the white cloudy precipitation floating. Does it affect the result of co-IP?
Thanks in advance.
Hi, I regularly work with HEK293T cells and I am also lysing them using NP40 (actually NP40 substitute). We use an amount of NP40 that ranges from 0,55% to 2% in a 50 mM HEPES-based buffer @ pH 7.5 - 8.0 containing 150 mM NaCl, 1 mM DTT and 5 mM MgCl2. I do observed indeed some flocculate but not always. We perform the lysis for 20-40´ @ 4 °C under constant shaking conditions. It could be some DNA. If the amount of NP40 in relation to the amount of cells you have to lyse you may have disrupted some nuclei.
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