Last updated: April 8 2004
Ref; Maehama et al.
Anal. Biochem. 2000, 279, 175-176
Preparation of
Malachite Green Reagent:
Prepare 100ml, can
store filtered solution for up to 6 months at 4¡C. use fresh millipored water.
Combine
25ml of 4.2% (w/v) ammonium molybdate in 4M
HCl
with
75ml of 0.045%(w/v) malachite green solution
(BDH stock solution is 0.5%, 9ml in 100ml -> 0.045%).
Stir for at
least 30 minutes (donÕt
miss this out, it is important), filter through 0.22µm syringe filter.
You get a green
solution which on filtering is amber.
Add Tween, 0.01%
v/v, to an aliquot
just before use.
Lipid
phosphatase assay:
The original assay
buffer used by the Dixon lab is
50mM Sodium
Acetate,
25mM bis.Tris
25mM Tris (pH 6.0)
2mM DTT
we use
20mM HEPES (pH
7.2)
100mM KCl
1µM ZnCl2
2mM DTT (added
fresh)
For long chain lipids:
Add lipid stock
solutions to a 1.5ml eppendorf tube. Add 5µl lipid stock (2500pmol) per experimental
point, 1 tube per point. Remember PI lipids are expensive. We normally use
a configuration of pure PI lipids, it is possible to supplement this with
an excess with more abundant lipids which would probably remove any artefacts
that may be due to lipid packing, but requires sonication or extrusion to
produce unilamellar vesicles.
Dry lipid
on the centrifugal vacuum dryer
(G11).
Resuspend in assay
buffer (50µl per point) using shaker for 20 minutes.
Add ENZYME (this
is normally a small volume relative to reaction volume e.g 5µl).
Incubate at room
temperature for given time with gentle shaking.
Stop reaction by
addition of 15µl of 100mM NEM- this is optional as IÕm pretty sure the
malachite green reagent stops the reaction.
Add 2 volumes of
malachite green solution per volume of assay supernatant.
Transfer 100µl to well of 96 well plate. Measure absorbance at 650nm using the plate reader in G07.
make
sure that you have control points. Best control is probably adding