Molecular Techniques and Methods

Isolation of Microsomes from Fungi

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Homogenization Buffer (100 ml)
10 mM Tris-HCl (pH7.4) ------------------------ 1 ml of 1 M Tris-HCl
10 mM KCl -------------------------------------- 1 ml of 1 M KCl
1.5 mM MgCl2 ----------------------------------- 150 ul of 1 M MgCl2
10 mM DTT -------------------------------------- 1 ml of 1 M DTT
0.5 M Sucrose ------------------------------------ 17 g
Add distilled H2O to make a final volume of ----- 100 ml
  • Autoclave and keep at 4oC.


    Resuspension Buffer (100 ml)
    100 mM HEPES (pH7.0) ------------------------- 10 ml of 1 M HEPES
    0. 05% Triton X-100 ------------------------------100 ul of 50% Triton X-100
    0.5 M Sucrose ------------------------------------ 17 g
    Add distilled H2O to make a final volume of ----- 100 ml
  • Autoclave and keep at 4oC.




    PROCEDURES

  • All steps should be performed on ice.

    1. Collect fresh cultured micellium by Whatinan No. 4 filter paper using suction flask.

    2. Transfer 10-50 g filtered micellium into a 350 ml Bead-Beater chamber (Biospec Products, PO Box 722, Bartelsville, OK74005).

    3. Add 160 ml Glass Beads (425-600g, Sigma G-8772) and fill to the rim with 10 - 15 ml of Homogenization Buffer. Add 1.5 ml of 10 mM PMSF.

    4. Homogenize 30 seconds in ice. Repeat 4 times. Always keep in ice to prevent overheat.

    5. Centrifuge filtrate at 800g for 10 min at 4oC to remove cell debris, starch and
    nuclei.

    6. Transfer supernatant to an ultracentrifuge tube.

    7. Centrifuge in a fixed-angle rotor at 65,000-70,000 rpm for 1.5 - 2 hour at 4oC.

    8. Collect the microsome pellet on the side wall with 500 ul Resuspension Buffer.

    9. Centrifuge 10 min at 800g and 4oC to remove cell debris, starch and nuclei.

    10. Carfully transfer supernatant to a new microfuge tube.

    11. Keep microsomes at -80oC for 6 months.




    NOTES




    KIT INFORMATION




    REFERENCES


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