Biology Reference
In-Depth Information
Biological question
Extract proteins
from organism
Fractionate by SDS-PAGE
Cut each lane into
16 equal fractions
Perform in-gel digestion
Extract peptides from gel
Analyse peptides by LC-MS/MS
Perform database search to
match spectra to peptides
Quantitate relative
abundances of proteins
Qualitatively explore
the proteome
Map proteins to
biological
pathways and
processes
Biological conclusion
Fig. 1
Schematic diagram of SDS-PAGE shotgun proteomics workfl ow
3.1 Protein
Extraction Protocol
1. The midsection of rice leaves were harvested from rice (
Oryza
sativa
cv. Nipponbare) plants grown in a greenhouse.
2. The leaf material was ground into a fi ne powder under liquid
nitrogen with a chilled mortar and pestle (
see
Note 1
). After
the nitrogen is evaporated from the powder (it should
appear dry), approximately 100 mg aliquots of the fi ne leaf
powder were weighed out into prechilled tubes using a
chilled spatula. The leaf material can be stored at −80 °C if
not used immediately.
3. The ground leaf material plus 2 mL of extraction buffer (8 M
urea in 100 mM Tris-HCl, pH 8.5, with 1 % (v/v) Triton
X-100) was subjected to probe sonication on ice for 3 × 20-s
pulses.
4. The extract was centrifuged at 17,000 ×
g
for 10 min at 4 °C
and supernatant removed to a clean tube (
see
Note 2
). Repeat
if required to remove all debris from the extract.
