1) Seed culture Grow 2 ml YPAD cultures of each strain at 30°, overnight to staionary phase.
2) Induction Centrifuge 1 ml of each culture, drain supernatant and resuspend pellet in 1 ml sterile H2O by vortexing.
Centrifuge again and dilute pellet 1:200 to 1:100 in 4 ml of induction medium (depending on how much the cells are expected to grow - cells should be in mid-log phase at end of induction. Grow at indcution temperature for 4-6 hours.
3) Lysis Centrifuge culture bubes and drain supernatant. Resuspend in 259 µl Thorner buffer. Add cell suspension to 2 ml cryovials. Add 100 µl of 500 micron glass beads.
4) Heat at ~ 100° for 3 min.
5) Mix cryovials on Bead Beater at 4°, 4 times for 1 min each, with 1 min. rest in between. Check for lysis under microscope - should be at least 80% lysed. If you don't have a Bead Beater, you can vortex at 4° for 20 min. (Saccharomyces) to 2 hr (Candida). Accept 50% lysis.
6) Add 500 µl H2O, 200 µl 5x non-reducing sameple buffer and 50 µl B-mercaptoethanol (now 1/4x thorner, 1x reducing sample buffer).
7) Heat at ~100°, 4 min. Centrifuge in cold-room 6min.
8) Add 5 µl of supernatant to 500 µl H2O and read A280 on spectrophotometer.
Thorner buffer 100ml
40mM Tris pH 8 4 ml of 1 M Tris pH 8
5% SDS 5 g of SDS
8M Urea 48g Urea
100 uM EDTA 20 µl 500mM EDTA, Add water to 100 ml final
Non-reducing Electrophoresis Sample Buffer
(ala Mark - a slight variation of the Bio-Rad formula)
Upper gel buffer 10 ml
Glycerol 25 ml
Bromphenol blue 12.5 mg
SDS 5 g
ddH2O add to 50 ml
Let glycerol drain from pipet for ~5min., add upper gel buffer, mix. Add bromphenol blue and SDS, mix. Heat slightly in microwave to get into solution. Add H2O.
For reducing buffer, dilute into protein sample to 1x, then dilute B-mercapoethanol 1:20 into sample.
(Upper gel buffer isL 0.5 M Tris pH 6.8 / 0.4% SDS)