Sucrose Gradient Fractionation for Subcellular Localization

Gradient Preparation without Mg+2

  1. Make STE solutions of 10, 20, 35 and 60% sucrose.

    • 10mM Tris pH7.6
    • 10mM EDTA
    • 10, 20, 35, or 60% weight to volume of sucrose.

  2. Layer the solutions sequentially to form a 5 ml step gradient.

  3. Tip the gradient to a horizontal position and let the sucrose diffuse for 5 hours.

  4. Alternatively, use a gradient pouring devices to make an instant linear gradient from STE60 and STE10.

Gradient Preparation with Mg+2
  1. Make STMg solutions of 10, 20, 35 and 60% sucrose.

    • 10mM Tris pH7.6
    • 2mM Mg+2
    • 10, 20, 35, or 60% weight to volume of sucrose.

  2. Layer the solutions sequentially to form a 5 ml step gradient.

  3. Tip the gradient to a horizontal position and let the sucrose diffuse for 5 hours.

  4. Alternatively, use a gradient pouring devices to make an instant linear gradient from STMg60 and STMg10.

Lysate Preparation
  1. Grow 100 ml of cells in YPD to an O.D.(600) of 0.5 to 1.0.

  2. Add sodium azide and potassium fluoride to 10 mM.

  3. Chill the cultures briefly on ice.

  4. Harvest cells by centrigugation.

  5. Wash once with 10 mM sodium azide, 10 mM potassium fluoride, 5 mM Tris (pH 7.6).

  6. Resuspend the pellet in 500 ul of STE10 plus protease inhibitors from 50X stock.

  7. Add glass beads to the meniscus, then vortex vigorously for 2 minutes.

  8. Add an additional 1 ml of STE10 or STMg10, then transfer the supernatant to a new tube.

  9. Clarify the lysate by spinning at 300 g for 3 minutes. This removes unbroken cells and cell wall debris.

  10. Layer 300 ul of the cleared lysate on top of a pre-made 5 ml sucrose gradient.

  11. Spin at 29,000 rpm (100,000 x g) in an SW55.1 rotor at 4oC for 12 to 18 hours. Or 33,700 rpm (132,000 x g) in an SW55.1 rotor at 4oC for 2.5 hours. Or probably anything in between.

  12. Carefully collect 380 ul fractions from the top of the tube with a micropipette.

  13. Dilute fractions to 1 ml with water, and then precipitate by the addition of 100 ul of 100% TCA.

    1. Vortex well then hold on ice for at least 20 minutes. Some papers suggest an hour.

    2. Spin in a cold (4oC) microcentrifuge for 5 minutes.

    3. Wash in 1 ml ice cold acetone:

  14. Vortex hard.

  15. Spin at full speed for 5 minutes to precipitate.

  16. Aspirate the acetone carefully and air dry pellets in a speed vacuum if available.

  17. Resuspend in 50 ul 1X sample buffer and load on the gel.