Marker enzyme for crude yeast membrane fraction?

Fergus Doherty Fergus.Doherty at nottingham.ac.uk
Wed Sep 3 08:57:03 EST 1997


I am developig a protocol for a simple subcellular fractionation of yeast
for an undergradate class and I need a simple microtitre plate based assay
for a particulate enzyme marker.  There are only 3 fractions to assay, a
1000g 5min pellet, and 100,000g 60 min pellet and a 100,000g supernatant. 
I have tried alpha mannosidase (vacuolar) without success, even using a lot
of protein.  (I assume p-nitrophenol alpha-D-mannopyrannoside, Sigma N2127
is the right substrate?).  Can anyone suggest an alternative.  It can be
localised to any membrane as long as it pellets readily.  I don't want to
use NADPH-cytochrome c reductase as this requires a 550nm filter, which I
don't have.  Same goes for cytochrome c oxidase.  How about succinate
dehydrogenase?  Unfortunately I think that there are low levels of SDH in
cells grown in glucose.

Anybody any ideas?

-- 
Fergus Doherty,
Dept Biochemistry,
Nottingham University,

Fergus.Doherty at nottingham.ac.uk
0115 970 9366 (74-41366 internal)



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