yeast cell extract - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

Glassbead Yeast Cell Extract for activity measurements

from CURRENT PROTOCOLS IN MOLECULAR BIOLOGY

http://www.geguchadze.com/PDF/protocols/CPonline/Toc/15848-20034.html

http://www.geguchadze.com/PDF/protocols/CPonline/Doc/17955-17955.html

Cell Disruption For Soluble Protein Using Glass Beads

These procedures are for small-scale preparations using a vortexer.

  1. Grow and harvest yeast cells as described in steps 1 to 4 of the basic protocol for spheroplast lysis. Determine the packed cell volume.

All subsequent steps should be carried out at 4°C.

  1. Resuspend cells in 1 vol glass bead disruption buffer. The cells can be frozen by slowly pouring this suspension into a plastic beaker filled with liquid nitrogen. Use enough liquid nitrogen to submerge the frozen paste. This frozen “popcorn” can be stored at −80°C. Thaw overnight on ice before proceeding.

  2. Mix cell paste with 2 vol glass bead disruption buffer.

  3. Add 4 vol of chilled, acid-washed glass beads.

For packed cell volumes of <10 ml:

  1. Transfer the cell suspension to an appropriately sized screw-cap centrifuge tube. The suspension should occupy no more than 60% to 70% of the capacity of the tube.

  2. Vortex the suspension at maximum speed for 30 to 60 sec at 4°C; place tube on ice for 1 to 2 min. Repeat 3 to 5 more times. Check the amount of cell breakage by visual inspection under a microscope. Proceed to step 7.

  3. Allow the glass beads to settle out and decant the supernatant.

  4. Add 2 to 4 vol glass bead disruption buffer to the glass beads and invert the tube 5 to 10 times. Allow the beads to resettle and decant the supernatant. Pool the super- natants.

  5. Centrifuge the pooled supernatants 60 min at 12,000 × g (SS-34 rotor at 10,000 rpm)

4°C. Collect the supernatant which represents the crude extract. For long-term storage, aliquot into small tubes, quick-freeze the tubes in liquid nitrogen, and store at −80°C.

Needed:

Chilled, acid-washed glass beads (0.45 to 0.55mm)

Glass bead disruption buffer 20 mM Tris⋅Cl, pH 7.9 10 mM MgCl2 1 mM EDTA 5% glycerol 1 mM DTT 0.3 M ammonium sulfate 1× protease inhibitor mix (see below) 1 mM PMSF The concentration of ammonium sulfate in the buffer can be varied between 0.1 and 1.0 M. Final concentrations above 0.25 M strip specific DNA-binding proteins and histones off chromatin and are therefore useful in obtaining factors that interact with nucleic acids. KCl and NaCl can also be added to final concentrations between 0.1 and 2.0 M.

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