Make a 200ul reaction mix containing: Stock Amount
0.1M Tris-HCl, pH 7.5 1M 20µl
1mM CaCl2 100mM 2µl
5mM Mannitol 500mM 2µl
/- 1% TX-100 10% 20µl
0.1mM substrate 10mM 2µl
Add 10-50ul of sample (I use 50ul of sucrose gradient fractions)
Vortex and place at 30oC for 1 hour
Boil 5 min to stop reaction
Let cool to room temperature
Spin 5 min in epp. microfuge
assay supernatant for liberated product by measuring emission in
fluorometer at the following wavelengths:
ex. 380 nm
em. 460 nm
Substrate is Boc-Gln-3-Arg-MCA purchased from Peninsula Laboratories.
Liberated product is aminomethyl coumarin. A 10mM stock is made in
DMSO and stored at 4oC in dark. For a 5 mg vial, add 698µl of DMSO.
When measuring product may have to dilute with 1.9ml of water to read
in cuvette. References are:
Cunningham and Wickner, Yeast, 5: 25-33 (1989)
Julius, D. et. al. Cell, 37, 1075-1089 (1984)
Determine latent KEX2 activity by subtracting values for /- TX-100.
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