Standard extraction of cells
N.B. Cells should be ideally 80-90% confluent when extracted. If you’re using
tumor cell lines like U2OS you may be able to get away with less confluent cells
by adjusting the lysis buffer accordingly.
1. Remove cells from incubator. Aspirate medium gently. Wash 1x with cold
PBS. Aspirate gently. Add 5 ml cold PBS and scrape cells (use the same
cell scraper but wash it in Milli-Q water in between plates). Transfer to a
15 ml Falcon tube.
2. Spin in the tabletop centrifuge 1000 rpm 5 min at 4˚C
3. Remove the PBS by gentle aspiration. Be careful not to get too close to
the cell pellet. Add TEB lysis buffer supplemented with protease
inhibitors. If you’re going to probe for a phospho-protein you’ll also need
phosphatase inhibitors. For protease inhibitors you can either add one
tablet from Roche to 10 ml TEB. This supplemented lysis buffer is stable
up to 2 weeks in the fridge. However, if you’ve got a small amount of lysis
buffer it makes more sense to add inhibitors individually. Add 10 µg/ml
each of pepstatin (1:100), leupeptin (1:1000) and aprotinin (1:1000) and
0.2 mM PMSF (1:500). For phoshatase inhibitors, add 10 mM NaF (1:50),
1 mM Na3VO4 (1:100) and 0.1 µM microcystin (optional, 1:10,000). Note
that microcystin is a toxin, so be cautious.
4. The lysis volume is typically 500-1000 µl for U2OS lines and 150-200 µl
for BJ/tert cells, since these typically yield less protein.
5. Add the supplemented lysis buffer to the cells and pipet up and down.
Transfer to an eppendorf tube on ice and extract for 15 min. Make sure
the micro-centrifuge is pre-cooled.
6. Spin in a micro-centrifuge at 13,000 rpm for 10 min at 4˚C. Save
supernatant (lysate) by transferring to another tube and store at -80˚C until
needed.