Western Blot

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 50uL of betamercaptoethanol (reducing agent) to 950uL of Laemmli dye.
2. Add Laemmli dye + betamercaptoethanol to lysate so that it dilutes to 1x (equal volumes of Laemmli and lysate if Laemmli is 2x).
3. Transfer to heat block set to 98C and boil for 5 minutes. 
4. Store at 4C (reboil if reloading).

BCA Assay (to check protein concentration)

1. Fill this in!

Running an SDS-PAGE gel

1. Use BioRad gel.
2. Load gel into gel running machine.
3. Add 1000 mL of running buffer to both the inside and outside compartments. Make sure the buffer covers the top of the wells.
4. Add 5uL of ladder to the first and last wells of the gel.  
5. Add 16uL of sample to each well. 
6. Run gel at 200V for 30 minutes (or until the dye front has reached the bottom of the gel. 

Running an SDS-PAGE gel

1. Use BioRad g

Lysis Buffer 

NP40 

0.1% tween 20

1x HALT protease inhibitor 

150 mM NaCl

50uM Tris 

Running Buffer