Pulse-chase Assay

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

  2. Harvest cells by centrigugation.

  3. Resuspend cells in minimal media with all the required amino acids, except methionine (or other amino acid of choice), to a final O.D.(600) of 1.0.

  4. Put cells in 15 ml snap top tubes and grow for at least 15 minutes with shaking

  5. Add 35S-methionine (or other amino acid of choice) label at 100 uCi per 0.5 O.D.(600) units.

  6. Incubate for desired time of pulse, usually about 10 minutes.

  7. Chase radioactive label by adding 5 ul per 500 ul of cells of cold cysteine/methionine mix (5 mg/ml each).

  8. Lyse zero time point as follows:

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

    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 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 immunoprecipitate 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 Lyse each withdrawn time point as in step (8).

  3. Add 1.0 ml IP buffer to each collected and lysed time point.

  4. Add 10 ul antibody to each sample, and incubate at 4oC overnight.

  5. Add 100 ml of 10% ProteinA-Sepharose to each sample, and incubate at room temperature for 2 hours.

  6. Spin samples at 1000 rpm in microcentriguge for 15 seconds, then carefully remove lysate and save in a new 1.5 ml eppendorf tube.

  7. Wash ProteinA-Sepharose beads once with IP buffer and twice with wash buffer by resuspending beads in buffer, incubating 1 minute and microcentrifuging at 1000rpm for 15 seconds. Apsirate the supernatant from the beads.

  8. Add 50 ul SUME buffer + 0.005% bromophenol blue to ProteinA-Sepharose beads and incubate at 65oC, or other desired temperature, for 10 minutes.

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

  10. After running gel, incubate in 10% acetic acid/25% isopropanol for 30 minutes.

  11. Rinse gels thoroughly and incubate in Amplify for 15-30 minutes.

  12. Incubate gel for 5-10 minutes in 2% glycerol.

  13. Dry gel and expose to film for desired period of time.

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

This is used for lysing yeast at pH6.8. For pH8 lyses, use the variation of SUME with Tris buffer known as SUTE.

Immunoprecipitation (IP) Buffer
(15 mM Na2HPO4, mw 142; 150 mM NaCl, mw 58; 2% Triton X-100, 0.1% SDS, 0.5% DOC, 10 mM EDTA, 0.02% NaN3)

TO MAKE 100 ml:

  • 0.213 g Na2HPO4
  • 0.87 g NaCl
  • 2.0 ml TritonX-100
  • 0.1 g SDS or 1 ml 10% SDS stock solution
  • 0.5 g deoxycholate
  • 2.0 ml 0.5M EDTA stock solution
  • 0.2 ml 10% NaN3 stock solution (optional)

Final pH should be 7.5. Sterilize through a sterile filter if azide (NaN3) is not added.
Wear a mask while handling the SDS. Requires Heat to get SDS into solution.

Wash Buffer
(50 mM NaCl, mw 58; 10 mM TRIS, mw 121; 0.02% NaN3)

TO MAKE 100 ml:

  • 0.29 g NaCl
  • 0.12 g Tris
  • 0.2 ml 10% NaN3 stock solution (optional)

Final pH should be 7.5. Sterilize through a sterile filter if azide (NaN3) is not added.

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.