Cells not lysing efficiently in hypotonic buffer. Help!
I am fractionating some mammalian cells (a tumour cell line) to obtain nucleus/debris pellet (800g), crude mitochondrial pellet (10000g), microsomal pellet (100000g) and the post-microsomal supernatant. However, I find that the hypotonic lysis buffer I use probably does not lyse the cells effectively (in fact, the lysis seems really poor), in spite of me incubating harvested cells in it for half an hour, followed by passing several times through a 26-gauge needle. This I know, because most of my protein is found post-lysis in the 800g pellet (probably unlysed cells). My protein is not found in the nucleus inside cells.
The lysis buffer I use is 20M Tris, 5mM MgCl2, 5mM CaCl2, 1mM EDTA, DTT, + protease inhibitors. What do I do to lyse cells effectively? I currently scrape cells with a rubber scraper. Will trypsinisation give better results? Some protocols call for Dounce homogenizers, but I currently don't have any. I have a Potter-Elvejhem homogenizer, though, but the volume of the homogenizer is a bit high (5 mL). Are Dounce homogenizers better than Potter-Elvejhem for hand-held lysis of cells? I can't find this information easily.
What is a foolproof method to ensure near-complete cell lysis, keeping nuclei and organelles intact?