PROTOCOL FOR CELL COLLECTION
1. Thaw out ingredients for lysis buffer and make PMSF. Once thawed,
prepare lysis buffer adding all ingredients EXCEPT PMSF! Do not
add PMSF until right before you are ready to use the lysis buffer!
Keep lysis buffer on ice.
2. You will need 3 sets of eppendorf tubes. 2 sets of 1.5ml (large) and
one set of .5ml(small) tubes. Label one set of large and the small set
with your treatment information and label one set of large tubes with
the cell line, treatment and date for storage.
3. Turn on the centrifuge and make sure you have cold PBS and clean
policemen.
4. To collect cells: Pour off media from plate and gently rinse 2x with
ice cold PBS. Tilt the dish and scrape downward with policeman. If
you don’t hear a scraping sound you are not scraping hard enough!
Pipette cells and put into the first set of large tubes and keep on ice.
Only collect 2 or 3 samples at a time!
5. Spin at 8000rpm for one minute.
6. Carefully vacuum off liquid leaving the pellet and put back on ice.
7. Add PMSF to your lysis buffer and invert to mix. Then add lysis
buffer to the pellet and pipette mix well. The amount of buffer you
add is dependant on the pellet size. (usually 40-150ul)
8. Let the cells lyse on ice for 10-15 minutes. Then spin them down at
12000rpm for 5 minutes. (while they are spinning, prepare an ethanol
bath: dry ice + 100% ethanol)
9. After the spin, pipette off the supernatant and put it into the second set
of large tubes. Take a 10ul aliquot and put into the set of small tubes.
This 10ul aliquot will be used for the protein assay. Freeze the
remaining lysate in the ethanol bath for 5 minutes and then store at –
80.