Biology Reference
In-Depth Information
14. These washing steps are critical to eliminate contaminant
proteins from the Digestion Medium such as the degrading
enzymes. Keep an aliquot of Digestion Medium and analyze its
content by SDS-PAGE to evaluate the level of contamination
in the cytosolic fraction.
15. Overlaying the extract with fl oating mediums II and III,
requires extreme care as the interfaces between the two
solutions can be easily disrupted. To avoid this, we recom-
mend overlaying the two mediums at 4 °C by placing a tube
at an inclined angle on ice and directly above it, the needle
of an upright syringe attached to a stand making contact
with the upper inside wall of the tube. Floating medium II
is placed in the syringe body, which is then slowly trans-
ferred into the resting tube by gravity. Repeat this step with
fl oating medium III. Note that this is important to achieve
a sharp interface.
16. The intensity and number of strokes may need to be opti-
mized. We found that three to six strokes results in a good
quality fraction, while more than six may provoke disruption
of organelles and result in cross-contamination.
17. The goal of this step is to remove chloroplasts and unbroken
protoplasts. The 85 % Percoll cushion prevents chloroplast
breakage during centrifugation, which would result in the
contamination of the middle phase. The objective of the top
layer (FMIII) is to maintain unbroken protoplasts on the top
interface.
18. Percoll gradients must be freshly made. Add the Percoll
Solution and Gradient Mixture in a separate tube to achieve
the desired Percoll Solution fi nal concentration.
19. The intact chloroplasts should sediment at the 45/85 %
(v/v) Percoll interface. The layer between the Chloroplast
Buffer and the 45 % (v/v) phase mostly consists of broken
chloroplasts.
Acknowledgements
This work (J.I., J.L.H., H.V.S. and Y.V.) was part of the DOE
Joint BioEnergy Institute (
http://www.jbei.org
)
supported by the
U.S. Department of Energy, Offi ce of Science, Offi ce of Biological
and Environmental Research, through contract DE-AC02-
05CH11231 between Lawrence Berkeley National Laboratory
and the U.S. Department of Energy. B.J.P. and G.E. are supported
by the ARC Centre of Excellence in Plant Energy Biology
(CE0561495).
