Could you recommend an efficient but mild way for lysis of sf9 cells for further protein purification? My protein is likely to be either cytoplasmic or nuclear in sf9 cells (knowing that it is a mostly nuclear mammalian protein). I usually deal with E. coli, for which sonication is a good lysis method, but I don't know if this can be applied to sf9 cells, and notably if the duration and strength of the sonication need to be modified for sf9 cells. Could anyone give me advice?
For a cytoplasmic preparation: baculovirus infected insect cells in medium were centrifuged at 2000g, washed with ice cold wash buffer [25 mM Tris/HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA], re-centrifuged at 2000g, and the pellet was then resuspended in ice-cold lysis buffer [25 mM Tris/HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 0.5% Nonidet P40, 4 mM 2-mercaptoethanol and 10 mM PMSF] and then incubated on ice for 10-15 min. The sample was then centrifuged at 6000g for 20 min. and the supernatant collected. Note the post-lysis pellet will look about the same size as the pre-lysis pellet since the nuclei are the majority of the volume of a cell by 60-70 hr post infection.
A nuclear prep. will need harsher conditions, see Redinbo and Staker papers on human topoisomerase I X-ray structures for a nuclear prep. for insect cells.
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