Alpha Factor Synchronization

This method includes practical tips and is based on α-Factor Synchronization of Budding Yeast, L. Breeden, Methods in Enzymology, 283:332 (1997).

1.  Grow cells o/n in YEPD 30C.

  1. Use reasonably fresh plate of cells to get reproducible growth kinetics
  2. Do not use synthetic medium for arrest/release. It is OK to use synthetic medium for overnight culture if cells double at least 1x in rich medium before arrest.
  3. Use other temperature if necessary for expt.
  4. Consider starting (1 or 2) 4-fold dilutions to get good density

2.  Culture for arrest must be in early log phase of growth. A600 is OK approximation of density, but not comparable across specs or across strains. Still, we target A600=0.2-0.3 before arrest.

  1. If A600 < 0.2, allow cells to continue growing to 0.2
  2. If 0.2 < A600 < 0.6, dilute cells in fresh medium to 0.2-0.3 and arrest
  3. If 0.6 < A600 < 0.8, dilute cells in fresh medium and allow to replicate 2-3x before arrest
  4. If A600 > 0.8, discard culture and start again.

3.  Add α-factor to 1x.

  1. 200x stock is 1mg/ml (~600μm ) of NH2-WHWLQLKPGQPMY-COOH in MeOH, stored at –20C.

4.  Monitor synchrony after one generation (90-120 min.)

  1. Look for 100% unbudded cells; most will form shmoos
  2. Sonication will help distinguish large budded from unbudded cells
  3. Mutants may require longer arrests

5.  Remove cells from α-factor and rinse.

  1. Filtering may be fastest and easiest; use just 5ml of new medium to rinse, don’t let cells get sucked dry. Don’t overwhelm your filter or vacuum set up with too many cells. We use the house vacuum and routinely filter 250mL cells OD 0.5 through a 0.45mm Millipore filter.
  2. Spinning to pellet cells is possible, too. In this case, rinsing may be skipped.

6.  Resuspend cells in warm fresh medium.

  1. Cells may be resuspended in ½ - ¼ lower volume than original culture.
  2. Shake culture vigorously to disrupt clumps.
  3. Samples are usually collected for 2 cell cycles (~180 min).