Cell Lysis

1. Aspirate media.
2. Add 500 uL of cold PBS to each well.
3. Aspirate.
4. Add 200 uL of trypsin.
5. Incubate for 90 seconds.
6. Neutralize with 800 uL of cold complete media.
7. Pipette up and down to disperse clumps.
8. Transfer contents of each well to 2 mL labelled eppendorf tubes.
9. Centrifuge/aspirate.  How long?  How fast?
10. Add lysis buffer.  How much?
11. Incubate at 4C for 30 minutes with constant agitation.
12. Centrifuge at 12000 rpm for 20 minutes.  Long/fast?
13. Remove tubes; place on ice.
14. Transfer supernatant to a new tube (on ice).  *At this point you can centrifuge to concentrate if necessary. - protocol?

Bradford Assay (to check protein concentration)

1. Fill this in!

Denaturing Protein from Lysate

1. Add Laemmli 2x to lysate (equal volumes of lysate and Laemmli if Laemmli 2x).
2. Transfer to heat block set to 98C and boil for 5 minutes.
3. Store at 4C (reboil if reloading).

Running an SDS-PAGE gel

1. Add gel to gel machine.
2. Add running buffer to cover wells on inner compartment.
3. Add running buffer to the outer compartment.
4. Add protein ladder to first well. How much?
5. Add around 25 uL of lysate (or appropriate volume according to lysate concentration) to each well.
6. Run gel at (voltage/amps).  How long?
7. Wash?
8. Stain with coomassie blue.