Biology Reference
In-Depth Information
8. It is important for effective disruption of protoplasts, that a
vacuum is formed with each stroke by pulling the Tefl on pestle
to create an air bubble in the solution under the pestle.
Generally, homogenizers with a space of approximately
50-100
m between the Tefl on pestle and the tube are the
most effective for disrupting protoplasts from
Arabidopsis
cells, with minimal disruption of organelles. To assess the
effectiveness of protoplast disruption with a given Potter-
Elvehjem homogenizer, take aliquots of protoplast samples
before and after homogenization and observe them using a
light microscope. Successful disruption of protoplasts in the
post-homogenized sample will be apparent by the presence of
released cellular contents (Fig.
4
). If this is not the case, then
adjustments to factors (
see
Notes 1
and
2
) may be necessary.
9. Isolation of the cytosolic fraction should be performed as
quickly as possible while maintaining a temperature of 4 °C
and preventing prolonged periods of incubation between
centrifugation steps. The time between homogenization and
isolation of the cytosolic fraction signifi cantly affects sample
integrity and yield.
10. The removal of Homogenization Buffer from the isolated
cytosolic fraction is critical as its constituents are not
compatible with protein precipitation or digestion, and
downstream LC-MS/MS analysis. We have investigated the
following methods to remove the homogenization buffer
from the cytosolic protein fraction: dialysis, size exclusion
FPLC, C4 hydrophobic interaction (C4) HPLC and
diafi ltration 5 kDa cut-off spin column. We found that the
diafi ltration spin column is the most effective method. It is
easy to use, has a high protein retention rate (~95 %) and is
compatible with LC-MS/MS. To remove most of the
homogenization buffer, the samples are concentrated,
diluted with 100 % H
2
O using a maximum spin column
volume and reconcentrated. This is repeated a total of three
times. Samples can then be used for analysis by immunob-
lotting or LC-MS/MS.
11. Preferably, place the seedlings in the dark for 24 h. This pro-
motes degradation of starch grana, which can break chloro-
plasts during centrifugation.
12. The seedlings do not need to be chopped. Vacuum infi ltration
is very effi cient to promote organ imbibition with the Digestion
Medium. Proper infi ltration will result in tissue turning very
dark green.
13. The Digestion Medium can be reused after removing
cellular debris by centrifugation (10 min at 3,000 ×
g
) and
storing at −20 °C.
μ
