Biology Reference
In-Depth Information
19. At a fl ow rate of 250 mL/h, plates will fi ll in approximately
17 min although shorter fi ll times will prevent sample warming
if a plate-cooling device is not available. Store plates at 4 °C
and work at 4 °C for steps 6 and 7 (Subheading 3.3 ).
20. Assuming that eight plates at approximately 1.5 mL per well
have been collected, pellets will be visible in all fractions above
0.15 A 280 (measuring 150
L at A 280 ); otherwise it may be
necessary to pool fractions. If collecting fractions individually,
one fraction from about fi ve plates will fi t into a standard
13.5 mL open ultracentrifuge tube. This approach will neces-
sitate multiple rounds of centrifugation for maximum yield
from 80 g FW of starting material.
21. A yield of approximately 60-80
μ
g of highly purifi ed Golgi
membranes split over 2-3 least contaminated fractions can be
obtained. Using 2-3 fractions gave over 80 % purity but Golgi
membrane yield can be increased substantially by pooling 6-8
fractions in experiments where an approximate 70 % purity is
acceptable. Obtaining highly enriched ER using this approach
is challenging given the associated migration of contaminat-
ing membranes (e.g., mitochondria and plasma membrane)
over these same fractions during FFE. Nonetheless FFE
migration patterns can be analyzed to distinguish ER and con-
taminating membrane patterns as was recently undertaken for
Golgi proteins [ 5 ].
22. Although this is a good method for determining organelle
migration, results are dependent upon the number of pro-
teomes available for a particular subcellular compartment, e.g.,
results for peroxisome migration will be more accurate than
those for ER migration. Therefore a low number of representa-
tive marker proteins is not always indicative of low abundance.
The accuracy of this method is a trade-off between the number
of markers used and the quality of markers.
μ
23. Organic solvents are incompatible with the standard tubing
used in FFE systems. Salt levels in pre-FFE samples should be
minimized as they will interfere with buffer conductivity.
Acknowledgments
This work conducted by the Joint BioEnergy Institute was supported
by the Offi ce of Science, Offi ce of Biological and Environmental
Research, of the US Department of Energy under Contract No.
DE-AC02-05CH11231.
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