MAKING YEAST EXTRACTS for WESTERNS

Tuesday, November 18, 2003

Description
MAKING YEAST EXTRACTS for WESTERNS

Procedure
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)

Recipes
TCA Buffer for 10ml

200ul 1M Tris 8
66.6ul 7.5M ammonium acetate NH4OAc
40ul 0.5M EDTA (important for protease inactivation)
9.7ml H2O
+ 200ul protease inhibitors : (ie to 2X final conc)
PMSF (stocks generally 100X)
Trasylol
Benzamide

TCA-Laemmli Loading Buffer
SOL I :
3.5ml 25% SDSor 4.375ml 20%SDS
3.5ml 100% glycerol 3.5 ml 100% glycerol
1.0ml 1M Tris base (not pHed)
spatula tip of bromophenolblue
-> H2O to 12ml 3.125ml H2O

SOL II :
200mM Tris Base(not pHed) -- 2ml of 1M
20mM EDTA -- 0.4ml of 0.5M
----> makes 10ml

SOL III :
1M DTT in H2O stored at -20 (1.543g/10ml)
mix:
480ul SOLI
400ul SOLII
120ul SOLIII --> gives TCA Laemmli buffer

Supplies

Tips

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MAKING YEAST EXTRACTS for WESTERNS