Biology Reference
In-Depth Information
4
Conclusion
Label-free shotgun proteomics based on SDS-PAGE fractionation
of proteins from rice is a simple, robust, and versatile technique.
The method can be used with proteins extracted from any tissue
type as varying extraction methods can be applied and data can be
collected for qualitative or quantitative purposes. In our experi-
ence, the technique is highly reproducible for the identifi cation
and quantitation of large protein data sets using spectral counting,
although other methods of label-free quantitation can be applied.
The range of achievable outcomes of label-free quantitative pro-
teomics in the fi eld of systems biology is bound to expand with
improvements in instrumentation and computer software.
5
Notes
1. During protein extraction, keep falcon tubes, scissors, and
spatula on dry ice. Wipe mortar, pestle, and spatula clean with
a lint-free tissue between samples.
2. Chill the centrifuge and rotor to 4 °C before starting protein
extraction.
3. DTT and bromophenol blue are not compatible with a BCA
assay and so were added after measuring protein concentra-
tion. SDS needs to be diluted to <5 % to be compatible with
BCA reagents.
4. Spray enough water on the plate to stop the gel drying out.
Wipe down all surfaces before starting with a lint-free cloth in
methanol, work in a hood if possible, and wear gloves and
sleeve protectors. Cover hair and beards, and take all available
steps to avoid keratin contamination.
5. It is not necessary to contain noticeable bands within a single
fraction; it is more important to make the gel fractions equal
sized and reproducible across replicates. This may be done by
dividing the gel into two fractions, and then each two fractions
into four fractions until 16 fractions are obtained. This may be
achieved by simply cutting each fraction down the middle for a
total of four rounds.
6. Treat gels stained with Sypro ruby, Deep Purple, or other fl uo-
rescent stains the same as a Coomassie-stained gel.
7. Allow trypsin to incubate with the gel pieces on ice for
20-30 min.
8. For the most complete extraction of peptides, peptides may be
extracted in sequential steps with the fi rst in 1 % (v/v) formic
acid, the second in 50 % (v/v) ACN, and the third in 90 %
(v/v) ACN (each diluted in water).
