Biology Reference
In-Depth Information
4. We stored birch pollen grains for more than 1 year on a lab
bench without any loss of allergens or changes in the allergen
release kinetics.
5. A solution of sucrose and boric acid is a weak buffer system and
usually has a pH around 5.6 at room temperature. However,
by addition of small crystals of MES or Tris the pH can be
adjusted. Whenever pH is an important parameter, use medium
M or add 10-25 mM MES to medium B and adjust with Tris.
6. The following stock solutions of the protease inhibitors were
prepared: 10 mM pepstatin A in DMSO, 10 mM leupeptin
in distilled water, 1 M PMSF in DMSO, and 1 mM E-64 in
distilled water.
7. Be sure that all chemicals used do not contain traces of phos-
phate as this will not only reduce the assay's sensitivity but can
also make the detection impossible. The phosphate reagent
will turn blue immediately after preparation! To remove traces
of phosphate from the glassware, all beakers, cylinders, mag-
netic stirrer bars, etc. were washed with methanol, followed by
1:5 diluted HCl and methanol and three times exhaustive rins-
ing with distilled water. We use plasticware which is also treated
as described above and used only for the phosphate reagents.
Never use phosphate-containing detergent for cleaning lab
ware when working on phosphate determination!
8. The following inhibitor stock solutions are used: 10 mM
sodium vanadate (Na
3
VO
4
) in distilled water, 15
M bafi lo-
mycin A1 in DMSO, and 100 mM sodium azide (NaN
3
) in
distilled water.
9. ATP may hydrolyze at acidic pH values and the released phos-
phate will decrease the test's sensitivity. Autohydrolysis of
ATP during the assay time is monitored in the substrate blank
cuvette.
10. Fresh lily pollen grains stick to each other by their pollenkitt.
One anther can be placed in 1 ml germination medium. Fierce
and intense shaking of the tube helps to wash the pollen grains
from the anther. The anther can be removed and the 1 ml pol-
len suspension can be transferred to a new tube. Washing with
5 % ethanol solution also helps to remove the pollenkitt and to
resuspend pollen grains, but may affect the germination rate.
11. Highest germination frequencies (>90 %) were obtained by
resuspending 30 mg lily pollen in 20 ml germination medium
M which were transferred to a large Petri dish (145 mm diam-
eter, Greiner, Kremsmünster, Austria). As a thumb rule, one
lily anther contains 12 mg (
μ
40,000) pollen grains.
12. We never succeed in germinating useful amounts of
Arabidopsis
pollen in liquid media for biochemical analyses.
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