Lissa1:Yeast Protein Extracts

From OpenWetWare
Jump to: navigation, search

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.