Biology Reference
In-Depth Information
so that the electrode buffers are not exhausted. For FFE
buffer 1 about 1.5 L is required for a run using the condition
described.
5. Cell suspension cultures are ideal for approaches such as
subcellular fractionation as a large homogenous starting mass of
tissue can be readily obtained. A fi ne suspension of Arabidopsis
cells provided the starting material for development of this
protocol. However, the same protocol can be applied to cultured
cells of other species and other sources of plant tissue. A frequent
source of failure when starting with tissue other than cell suspen-
sion cultures is insuffi cient starting material. Keep in mind that
yield is highest from suspension-cultured cells, for which 30 g
fresh weight after fi ltering is the minimum workable starting
amount for this approach.
6. Maintain approximately this ratio for other starting tissue
weights and buffer volumes. For different tissue sources,
researchers may want to use a lower homogenization buffer
volume: tissue weight and lower FW starting material:enzyme
ratios.
7. Rotate at the lowest possible speed at which cells remain in
suspension. Enzymes should be added to the digestion buffer
immediately before use. Enzymes are easily solubilized by
vigorous shaking in a 50 mL aliquot of digestion buffer, prior
to their addition to the main solution.
8. The pellet/protoplasts are delicate; decant the supernatant
gently to avoid breaking cells.
9. As a guide, the negative pressure on the upstroke should result
in an approximate 2 cm space or air bubble between the
plunger and the homogenate. For the fi rst few attempts at pro-
toplast disruption, checking the result using a light microscope
should reveal rupturing of at least 75 % of the protoplasts.
Intact plastids can be used as an indicator for intact Golgi
cisternae or other subcellular compartments. The key to this
step is ensuring enough mechanical stress to disrupt the proto-
plasts without destroying subcellular integrity. The number of
strokes of the homogenizer should be calibrated using light
microscopy.
10. Ideally, use about 20 g fresh weight starting material per
gradient. From four gradients expect 8-14 mL of protein at
0.8
L.
11. This step should result in a yellow-colored cushion about 2 mm
thick (assuming starting with 60-80 g fresh weight cells).
μ
g/
μ
12. Complete removal of the supernatant is a compromise between
the quality of step gradient formation and disturbance of the
cushion, with ensuing loss of yield.
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