Halo Assays
K. Kozminski (9/14/99)
1. Grow an overnight culture of your favorite mutant yeast strain and a wild-type
control.
2. Dilute to OD600 ~0.1. 1mL will be used per plate.
3. Pipette 1mL of diluted culture onto a plate. Drier plates work best (set out on
the bench the night before use.) Gently swirl the culture until the entire
surface of the plate is covered with medium. Tilt the plate and pipette off the
excess culture.
4. Leave the plates to dry for ~1 hr.
5. Onto sterile filter disks (DIFCO concentration disks, 1/4 " diameter) pipette
10µL of your drug/compound of choice. With sterile tweezers, drop the disk
onto the lawn of cells. Four disks fit nicely on a standard Petri plate – 3 disks
can be used for a serial dilution of the drug; the fourth disk should be used as
a solvent blank.
For Latrunculin A, use 0.5, 1 and 2mM dilutions.
For Alpha factor, use 0.5, 1 and 2mg/mL dilutions.
6. Incubate the plates for 1-2 days until a lawn of cells appear. Halos (regions of
no cell growth) should appear around the filter disks. Depending upon how
long the plates are incubated, double halos may appear, consisting of a ring of
no cell growth around the disk and an outer concentric ring with partial cell
growth. This outer ring consists of petites.
7. Measure the diameter of each halo on each plate where absolutely no cell
growth has occurred. If a double halo appears, measure the innermost halo
closest to the disk where no cell growth has occurred (this is the accepted
standard of what to measure, contrary to some but not all earlier Drubin lab
papers).
8. To calculate the relative apparent sensitivity of a strain to a given
drug/compound, plot a straight line of halo diameter vs. log of drug
concentration. Using the line equation, calculate the drug concentration
needed to produce a halo of x diameter. "Relative apparent sensitivity" is
calculated by dividing the drug concentration to produce a halo of x diameter
on an experimental plate by the drug concentration required to produce a halo
of the same x diamter on a wild-type/control plate.
See Figure 6 in Reneke J.E., K.J. Blumer, W.E. Courchesne, and J. Thorner.
(1988). The carboxy-factor receptor is a
regulatory domain. Cell 55:221-234.