Galactose Pulse-chase Assay

  1. Grow desired volume of cultures to 1 O.D.(600) (~2E7 cells/ml) using selective media with 2% raffinose instead of glucose.

  2. Add galactose to 2% final concentration, and incubate for desired time determined for optimal expression of galactose-inducible expression of your target protein, usually 30-60 minutes.

  3. After growth in galactose for desired pulse time, add glucose to 2% final concentration.

  4. Lyse zero time point as follows:

    1. Harvest 2.0 ml cells in a micro-centrifuge by spinning full speed for 3 minutes.

    2. Decant and discard supernatant.

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

    4. Add 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. Remove the lysate from the beads with a blue pipette tip to a new 1.5 ml eppendorf tube.

    7. Spin 5 minutes to clarify. Remove supernatant to a new 1.5 ml eppendorf tube.

    8. Store at 4oC until ready to run all time points.

  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 Lyse each withdrawn time point as in step (4).

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

  4. After running gel, transfer proteins to nitrocellulose and perform 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.


SUME BUFFER
(1% SDS, 8 M Urea, 10 mM MOPS, pH 6.8, 10 mM EDTA)

TO MAKE 100 ml:

  • 1.0 ml 1 M MOPS, pH 6.8 stock solution
  • 1.0 g SDS, or 10 ml 10% SDS stock solution
  • 48.05 g Urea
  • 2.0 ml 0.5 M EDTA stock solution

This is used for lysing yeast at pH 6.8. For pH 8 lyses, use the variation of SUME with Tris buffer known as SUTE.

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

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

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

LEUPEPTIN AND PEPSTATIN (5 mg/ml)

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


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