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solution lysis - (Apr/30/2002 )

hi everybody,

please, is there someone who can tell me which solution lysis can I use for yeast because I want to extract protein for Western blot after doing two hybrid system


I would suggets to use glass beads to extract proteins from yeast. Go to the Invitrogen web site ( and download Pichia pdf files. If you have troubles to get the protocol, e-mail me at:; I'll give you the detailed protocol.


Here is the protocol:
Harvest 5ml cells at OD = 1.0 in 15ml falcon tube[Optional : freeze pellet in liquid nitrogen]
r/s in 500ul cold TCA buffer w/ 2X Protease inhibitors
prepare tubes with glassbeads and 500ul cold 20% TCA
keep on ice
put cells in TCA tubes
bead-beat2X 30" at high setting, (keep on ice inbetween)
take liquid phase off with thin tip into new tube on ice N.B. don't spin yet)
wash beads with 500ul 1:1 TCAbuffer/20% TCA
beadbeat again 30" take liquid to eppendorf
spin 10' in coldroom
aspirate off s/n
r/s pellet in 300ul TCA-Laemmli buffer with inhibitors
boil 10'
spin 10' at RT, put s/n into fresh eppendorf
run 20ul (=1ug protein approx)