Biology Reference
In-Depth Information
3.2 Preparation
of Pollen Exudates
To identify allergenic pollen proteins, pollen grains are often
incubated in solutions to release the allergenic proteins. It has
to be noted that the fractions obtained by this method have to be
denoted as “pollen exudates” or “pollen diffusates” to distinguish
them from pollen extracts which are prepared by damaging the
pollen grains. Indeed, damage of pollen grains has to be avoided
when allergens should be identifi ed in subsequent proteomic
experiments because otherwise the high amounts of intracellular
proteins would cover the less abundant allergens.
1. Resuspend 100 mg pollen grains in 1 ml medium ( see Note 14 ).
2. Gently shake for 20 min at RT or use an end-over-end rotator.
3. Pellet larger pollen grains, e.g., lily pollen, by centrifugation at
3,000 × g for 15 min at RT. Collect the supernatant (=pollen
exudates [ 8 ]). Smaller pollen grains (e.g., pollen grains from
Arabidopsis , birch, mugwort) should be fi ltered through 5
m
centrifugal fi lter units (e.g., UFC30SV00, Millipore, Billerica,
MA, USA) using 1,000 × g and 5 min at RT.
4. Dialyze the pollen exudates against water or 50 mM NH 4 CO 3
and lyophilize the solution for long-term storage at −20 °C.
μ
3.3 Preparation
of Homogenates,
Cytosolic and
Membrane Fractions
from Pollen
The quality of the preparation and, thus, the quality of the investi-
gated proteins very much depend on performance during the
preparation steps. All steps should be performed in an ice bath
(0-4 °C) and speedily. The following procedure was optimized for
lily pollen to investigate the membrane proteome [ 1 , 9 ], but can be
adapted to other pollen species. The starting material is suffi cient to
allow organelle membrane isolations and to perform several electro-
phoresis and immunodetection assays.
1. Resuspend pollen grains from 25 fl owers in germination
medium or water. Pellet the pollen grain (1,000 × g , 15 min,
4 °C) and resuspend in ice-cold homogenization buffer.
2. Homogenize the pollen grains with a tefl on pistil (Potter-
Elvehjem type, Potter S from Sartorius, Germany) by circa 20
down- and upward strokes ( see Note 15 ).
3. Filter the homogenate through a 10-30
μ
m nylon mesh
( see Note 16 ). Keep the fi ltrate.
4. Centrifuge the fi ltrate at 7,500 × g , for 15 min, at 4 °C. Decant
the supernatant carefully into a high-speed centrifugation tube
( see Note 17 ). Try to keep the lipids which fl oat on top of the
medium in the tube.
5. Centrifuge the supernatant at 48,000 × g for 75 min at 4 °C to
pellet all membranes.
6. Decant the supernatant carefully without the fl oating lipids.
This supernatant represents the cytosolic fraction which contains
all soluble proteins including those from organelle lumens.
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