Day1:

• Plate yeast on EMM +A+T+(L or U), incubate @ 30oC
• Inoculate a 2ml culture using the same media overnight @ 30oC

• Place the cultures in 15ml falcon tubes and spin in Beckman for 3 minutes at 1500rpm.
• Remove the supernatant by placing a sterile pipet tip on the pasteur pippete used for suction and draw off the media carefully.
• Add 5ml of sterile water, resuspend and spin again, same speed and time.
• Wash with water 2 more times
• Finally resuspend the cells in 2ml (THE SAME VOLUME OF THE STARTING CULTURE) of media and dilute 1:100 into fresh media EMM +A +Lor U WITHOUT thiamine.
• Make 1 5 ml culture with thiamine as a negative control.
• shake for 20 hrs at 30oC

• Precool all equipment at 4oC
• Prepare a beaker with boiling water
• Spin samples @ 1500rpm 3min and discard the supernatant
• Add 5-10 ml Stop solution, resuspend, spin and discard the supernatant
• Add 20-100 ul RIPA buffer (depending on pellet size), PMSF (1000x), leupeptin (1000x), pepstatin (1000x) and aprotenin (100x) and resuspend
• Add the suspension to clean screw cap microfuge tubes
• Add 300ul of glass beads (or 0.5 mm zirkonia beads from Biospec)
• Place on FASTPrep (Speed = 6, Time = 20sec) or vortex 1 min
• Put samples back on ice
• Check a sample under the microscope to see if the cells were sufficiently lysed, if not repeat the FASTPrep step again.
• Estimate the volume of the suspension and add 2xLoading buffer accordingly (300 to 500 ul)
• Place in boiling water for 5min, spin in microfuge, fill supernatant into a new microfuge tube and store at -20oC
• Load 5 - 15 ul on SDS gel, run at 15mAmp until the dye reaches the separating gel, then increase to 25mAmp for 2hrs
• Make a Western Blot.