Biology Reference
In-Depth Information
Fig. 2
Proteins are extracted from (
a
) rice leaves, stems, or roots, or (
b
) rice cell cultures and separated by
SDS-PAGE. (
c
) After visualizing with a protein stain, (
d
) lanes are sliced into 16 equal fractions and transferred
to a 96-well plate (or microcentrifuge tubes) for in-gel digestion
100 % (v/v) ACN to dehydrate gel pieces. The samples were
vortexed during these incubations.
5. The ACN was removed and the gel pieces allowed to either
air-dry on bench or in fume hood for 10 min, or evaporated
briefl y in a vacuum centrifuge without heat. The gel pieces
should be noticeably shrunken and white.
6. The gel pieces were rehydrated with 50
L 10 mM DTT in
50 mM NH
4
HCO
3
and vortexed to mix. The tubes were
briefl y centrifuged and proteins were reduced for 60 min at
37 °C.
7. The gel pieces were cooled to room temperature, DTT solu-
tion was removed, and 50
μ
L of 55 mM IAA in 50 mM
NH
4
HCO
3
was added and vortexed to mix. The tubes were
briefl y centrifuged and proteins were alkylated for 45 min in
the dark at room temperature.
8. The IAA solution was removed and the gel pieces were washed
with 100
μ
L of 100 mM NH
4
HCO
3
for 5 min with vortexing,
and then washed twice with 50 % (v/v) ACN and 50 % (v/v)
50 mM NH
4
HCO
3
for 5 min with vortexing.
9. The gel pieces were dehydrated with 100
μ
L ACN as in
steps 4
and
5
. Again, the gel pieces should be noticeably shrunken and
probably white.
μ
