Biology Reference
In-Depth Information
13. Approximately 50 tobacco flowers are needed for 20 mg
pollen grains.
14. A large variety of solutions and media has been used for prepa-
ration of pollen exudates (water, germination media,
phosphate-buffered saline (PBS), or NH
4
CO
3
). Note that the
choice of solution often selects already the type of proteins that
are released [
8
]. Especially the ionic strength and the pH of
the solution affect the release of proteins from the pollen coat
because the cell wall acts as an ion exchange matrix.
15. The upward movement of the pistil creates a negative pressure
and causes the pollen grains to burst. Carefully move the pistil
without sucking too much air into the solution. Other ways of
breaking up pollen grains are possible, e.g., ultrasonication,
freezing in liquid nitrogen, and homogenization by pistil and
mortar. However, these methods generated a large number of
mixed vesicles which cannot be separated anymore by means of
marker enzyme analysis. Mixed vesicles occur during the
homogenization step by vesicle bursting followed by resealing
or fusion with other burst vesicles.
16. The fi lter retains all exine shell, partially broken pollen grains,
and some large organelles (nuclei, generative cells). Smaller
mesh width (5
μ
m) can be used without any severe loss of
material.
17. The pellet contains heavy organelles like starch grains, nuclei,
generative or sperm cells, and mitochondria.
18. Carefully resuspend the microsomal pellet on ice by pipetting
the centrifugation buffer over the pellet until it resolves by
itself. This can take up to 10 min! Never pierce into the pellet
with the pipette tip or try to mix the pellet with the pipette tip.
This will create large MF lumps but not a homogeneous frac-
tion which is important for reproducible results in the follow-
ing experiments, e.g., equal loading of SDS-PAGE gels, ATP
hydrolysis assays, or H
+
transport experiments.
19. It has to be noted that the sucrose step gradient has to be opti-
mized for every system to obtain optimal organelle vesicle sep-
aration. First experiments are run with a continuous sucrose
gradient and the distribution of organelle membranes is moni-
tored with marker enzyme assays, by proteomic identifi cation
of marker enzyme peptides, or by immunodetection of organ-
elle markers along the gradient. A step gradient centrifugation
is much faster than the centrifugation of a continuous gradient
(ca. 16 h) and the sucrose steps can be adapted to the special
needs of the experiments (e.g., separation of all organelles or
separation of one or two organelle fractions with a higher
purity). Parameters like the sucrose concentration, the volume
of the sucrose steps, the number of sucrose steps, and the cen-
trifugation speed as well as time have to be optimized.
