Cycloheximide-chase Assay

  1. Grow 10 ml cultures to O.D.(600) = 1.0 (~2E7 cells/ml) using selective media.

  2. Add 10 ul 50 mg/ml cycloheximide (dissolved in DMSO). Vortex to mix.

  3. Lyse zero time point as follows:

    1. Harvest 2.0 ml cells in a round bottomed eppendorf tube by spinning full speed for 3 minutes in the microcentrifuge.

    2. Decant and discard supernatant.

    3. Add 200 ul of SUMEB buffer + protease inhibitors.

    4. Add 1 100 ul scoop of 0.5 mm Acid Washed Glass Beads.

    5. Vortex in multivortexer for 3 minutes (speed setting 7 on our current machine).

    6. Incubate for 10 min at 65oC or whatever temperature is desired.

    7. Remove the lysate from the beads with a blue pipette tip to a new 1.5 ml conical bottom eppendorf tube.

    8. Spin 5 minutes to clarify. Remove supernatant to a new 1.5 ml conical bottom eppendorf tube.

    9. Store at 4oC until ready to load gels.

  1. Incubate remaining portion of culture at desired temperature and remove 2.0 ml aliquots at desired time points.

  2. Lyse each withdrawn time point as in step (3).

  3. Load 10-30ul samples onto a SDS-PAGE gel or dot blot.

  4. Analyze by western.

Note that protease inhibitors are added from stocks to give 50 fold dilution.
Example: 20 ul of stock per 1ml buffer.

Use IMMEDIATELY after adding protease inhibitors.


SUMEB BUFFER
(1% SDS, 8M Urea, 10mM MOPS, pH 6.8, 10mM EDTA, 0.01% bromophenol blue)

TO MAKE 100ml:

  • 1.0ml 1M MOPS, pH6.8 stock solution
  • 1.0g SDS, or 10ml 10% SDS stock solution
  • 48.05g Urea
  • 2.0ml 0.5M EDTA stock solution
  • 1.0ml 1% bromophenol blue

This is used for lysing yeast at pH6.8. For pH8 lyses, use the variation of SUMEB with Tris buffer known as SUTEB.
Also, bromophenol blue can be left out if dye is not desired, such as using the lysate for immunopreicipations.

50X STOCK Protease Inhibitors (store at -20oC)

PMSF (87 mg/ml)
([500mM] phenylmethylsulfonyl fluoride)

TO MAKE 30 ml:  Dissolve 2.61g PMSF in DMSO to equal a final volume of 30 ml.

LEUPEPTIN AND PEPSTATIN (5 mg/ml)

TO MAKE 10 ml:  Dissolve 50mg LEUPEPTIN or PEPSTATIN in DMSO to equal a final volume of 10 ml.

TPCK (5 mg/ml)
(tosylphenylalanine chloromethyl ketone)

TO MAKE 10 ml:  Dissolve 50mg TPCK in DMSO to equal a final volume of 10 ml.


* Note: The EDTA in the SUME buffer is also a protease inhibitor.