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PROTOCOL FOR ISOLATING CYTOSOLIC AND …
Christopher Scull November 2010 PROTOCOL FOR ISOLATING CYTOSOLIC AND mitochondrial extracts FROM mouse MACROPHAGES ( PROTOCOL is modified from the PROTOCOL provided by Qiagen) Use "Qproteome Mitochondria Isolation Kit" from Qiagen (catalog # 37612) 1) Grow macrophages in 6-well plate, with ~400,000 cells per well. Many cells are needed for this PROTOCOL . At a minimum, 3 wells from the 6-well plate must be combined for each sample. 2) At the end of the experiment, wash all of the cells once with HBSS. Add 1ml per well of HBSS and gently scrape the cells. Collect at least three wells into one 15ml-conical tube. Wash the wells again with 1ml HBSS and combine all collections in the same 15ml-conical tube. Pellet the cells by spinning @ 1500 x g for 5min 3) Be sure that Protease Inhibitors have been added to the Lysis Buffer. Resuspend the cell pellet in 1ml of ice-cold Lysis Buffer. Incubate for 10min at 4deg on an end-over-end shaker.
Christopher Scull – November 2010 . PROTOCOL FOR ISOLATING CYTOSOLIC AND MITOCHONDRIAL EXTRACTS FROM MOUSE MACROPHAGES (Protocol is modified from the protocol provided by Qiagen)
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