Lissa1:Yeast Protein Extracts

Solution 1:

• • 1.85 M NaOH
  • 7.3% B-mercaptoethanol
  • Must make this fresh!

Solution 2:

• • 50% TCA (store 4 degrees)

Solution 3:

• • Acetone (store -20 degrees)

Sample buffer:

• • 1.25 mL 4x Laemmli protein loading buffer
  • .25 mL 1 M DTT
  • .15 mL Tris base
  • 3.35 mL water
  • Either make fresh or store.

Steps:

1. Pellet 1.5 mL of cell culture having OD 1.5.
2. Remove supernatant.
3. Freeze in liquid nitrogen and store at -80 degrees.
4. Add 150 uL of Sol. 1 to each tube, vortex to resuspend.
5. Incubate 10 min on ice.
6. Add 150 uL of Sol. 2 and invert to mix.
7. Incubate 10 min on ice.
8. Centrifuge 2 min at 4 degrees, remove supernatant.
9. Rinse pellet with 1 mL of Sol. 3.
10. Centrifuge 2 min at 4 degrees, remove supernatant.
11. Dry tubes upside down under a tube tray on a paper towel for 3 min.
12. Add 50 uL sample buffer.
13. Break up pellet with tip. Vortex.
14. Boil in sand-boiler for 5 min, vortex again, load around 20 uL on sumo gel.