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solution lysis


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#1 mkl

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Posted 30 April 2002 - 07:18 AM

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
thanks


#2 er44

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Posted 30 April 2002 - 08:30 AM

I would suggets to use glass beads to extract proteins from yeast. Go to the Invitrogen web site (www.invitrogen.com) and download Pichia pdf files. If you have troubles to get the protocol, e-mail me at:
erodrigu@danone.com; I'll give you the detailed protocol.
er44

#3 er44

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Posted 30 April 2002 - 09:42 AM

Here is the protocol:
MAKING EXTRACTS for WESTERNS
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)






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