Biology Reference
In-Depth Information
of added inhibitors, vesicle fractions, substrate, etc. Add the
inhibitors, e.g., 200
M vanadate and 100 nM bafi lomycin for
measuring the azide-sensitive ATP hydrolysis. Add the specifi c
inhibitor to one-half of the cuvettes, in this example 1 mM
azide, to determine the difference of ATP hydrolysis activity in
the presence and absence of azide (azide-sensitive ATP hydro-
lysis activity,
see
Note 8
).
3. Add equivalent volume of the vesicle/interphase fractions to
achieve between 5 and 10
μ
g protein per cuvette. Mix well
with the pipette tip and incubate for 30 min at RT.
4. Start the reaction with 9
μ
l 100 mM ATP stock solution
(
see
Note 20
). Mix well and incubate the reaction for 60 min
(
see
Note 21
).
5. Stop the reaction by addition of 700
μ
l phosphate reagent.
Incubate for color development for 60 min and measure the
absorption at 820 nm.
6. The absorption value (
A
vesicle
) is corrected for blank values to
obtain the amount of liberated phosphate only by enzyme
activity as follows:
μ
(
)
−
(
)
AA A
=
−
−
A
−
A
A
−
A
corr
vesicle
bufferblank
vesicleblank
bufferblank
substrateblank
bufferblank
=
AA
−
−
A
strateblank
+
A
vesicle
vesicleblank
sub
bufferblank
The corrected absorbance value can be used to calculate
phosphate concentration using a calibration curve.
4
Notes
1. Mature anthers can be identifi ed by their exposed pollen
grains. Adjust the tube size to the amount of pollen grains you
need to store: e.g., lily pollen are best stored in 1.5 ml reac-
tion tubes for single anthers, 15 ml centrifugation tubes for up
to 5 fl owers, and 50 ml centrifugation tubes can be used to
store anthers from 25 to 50 fl owers. Sometimes, anthers can
be dried at room temperature in a desiccation vessel before
freezing.
2. To avoid exploding of the tube, pinch a small hole into the lid
and let the liquid nitrogen completely evaporate before closing
the lid. Wear protection gloves and goggles!
3. Keep in mind that most allergenic pollen grains originate from
wind-pollinating plants which means that you hardly collect
any pollen grains on a windy day. For best amounts of pollen
allergens, pollen grains should be dry. Otherwise they have
already lost most of their proteins from the pollen coat. Use a
pierced lid to close the tube. Always avoid humidity.
