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 2500 RPM for 8 minutes.
10. Aspirate supernatant. 
11. Add 500 uL of lysis buffer. 
12. Incubate at 4C with agitation for 30 minutes. 
13. Centrifuge at 12000 RPM for 20 minutes. 
14. Transfer supernatant to new labelled tubes and store at 4C (or on ice). 
15. Discard pellet. 
     

Denaturing Protein 

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

BCA Assay (to check protein concentration)

1. Fill this in!

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.

Lysis Buffer 

NP40 

0.1% tween 20

1x HALT protease inhibitor 

150 mM NaCl

50uM Tris 

Running Buffer