Plant Mitochondrial Proteomics - Plant Proteomics: Methods and Protocols - page 500

4. 0.2-1 % (w/v) bovine serum albumin (BSA) acts as an acces-
sible sacrifi cial protein target for released proteases and to
scavenge free fatty acids.
5. Reductant (cysteine, ascorbate, dithiothreitol (DTT), or
β -mercaptoethanol) is added freshly prior to homogenization

to a fi nal concentration of 5-20 mM to protect against oxi-
dants produced or released during homogenization.
6. For green tissues the addition of 1-2 % (w/v) polyvinylpyr-
rolidone (PVP-40) helps to remove phenolic compounds
liberated from the vacuole that can damage organelles in the
initial homogenate.
7. The ascorbate (Na L -ascorbate, Mr 198.11) and cysteine (Mr
137.7) are added to the chilled homogenization solution
immediately before use, correcting the pH after addition.
8. Available from Kinematica ( http://www.kinematica-inc.com/ ).

9. It is recommended to make up only about 10 mL of reaction
master mix and use for a series of eight to ten assays
immediately.
10. Aliquots of lysis and rehydration buffers are stored indefi nitely
at −20 °C.
11. TFA should be prepared at the concentration of 10 % (v/v)
and diluted to the required concentration before use.
12. Trypsin typically comes in aliquots of 25 μ g that is resuspended

in 250 μ L of 0.01 % (v/v) TFA (100 μ g/mL trypsin) and

g/mL and 10 mM
NH 4 HCO 3 using 200 mM NH 4 HCO 3 and water.
13. Saturated CHCA matrix solution can be used for 7 days; all
other solutions should be made immediately prior to use.
14. Many varieties of commercial columns with many variations in
column architecture and stationary phases exist; we typically
use homemade Microsorb C18 0.5 mm × 100 mm, 5

made up to fi nal concentration of 12.5 μ

μ m,

100 Å columns.
15. Many varieties of commercial columns with many variations in
column architecture and stationary phases exist; we typically
use Agilent Zorbax 300SB C18 75 μ m × 150 mm, 5 μ m, 300 Å

columns.
16. Available from Merck Millipore ( http://www.merckmillipore.
com/ ).
17. Available from Beckman Coulter ( http://www.beckmancoul-
ter.com/ ) .
18. Care must be taken when layering the washed mitochondria on
top of the prepared gradient solution to avoid disturbing the
gradient. This is best achieved by running the extract down the
side of the tube as opposed to adding it drop by drop.