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westerns

Kathy Shire shirek at FHS.CSU.MCMASTER.CA
Wed Aug 3 13:04:20 EST 1994


Hi Peter,

I have tried a number of methods.  The following is a bit crude.  It's a
good method to look for expression/no expression  of a protein but it's not
good for quantifying a protein because you can't do a Bradford Assay on
your lysate.  One good thing about it...it's really easy.

grow yeast O/N in 50 mL YEPD (this medium works well with stable plasmids)
such that OD600=0.5-1.0 the next morning.

(You can quantitate this method somewhat by spinning down the same # of cells)
on ice 10min
spin 4 C  3000rpm 5min in 50mL conical tube (Beckman)
resuspend cells in about 500uL ddH20 (ice cold) and transfer to eppendorf tube
spin a few seconds in microfuge and aspirate of supernatant
add 4X volume (of cells) of 1X Cracking Buffer (add ddH2O first)
ie for 100uL of cells
   resuspend cells in 320uL ddH2O
   add 80uL 5X Cracking Buffer
boil samples 8min
on ice 5min, spin in microfuge 4 degrees C 5minutes
transfer supernatant to new tube

For my protein (driven by ADH promoter), I load 25uL of lysate on a 1.5mm
mini-PAGE.  This gives me a good signal.

5X Cracking Buffer (any protein loading buffer will do)
5mL glycerol
2.42g Tris
2.5mL 1M DTT
2.5mL 20% SDS
3.5mL ddH2O
a dash of bromophenol blue
store at -20C (do not repeatedly freeze/thaw because SDS will PPTE out)

I hope this helps.

Kath






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