Biology Reference
In-Depth Information
2
Materials
2.1 Protein
Extraction
1. Shock-frozen plant tissue of interest (e.g., whole leaves and
roots,
see
Note 1
). Plan your experiment and the material
requirement for at least three to four independent biological
replicates [
9
] (
see
Note 2
).
2. Mortar and pestle.
3. Extraction buffer: 40 mM Tris-HCl, pH 6.8, 10 % (v/v)
glycerol.
4. Inhibitor cocktail for proteases (e.g., cOmplete, EDTA-free
Protease Inhibitor Cocktail, Roche, Basel, Switzerland).
5. 20 % (w/v) SDS.
6. 2 M Dithiothreitol (DTT).
7. 1 % (w/v) Bromophenol blue (optional).
8. Heating block.
9. Standard protein assay (e.g., Bradford, bicinchoninic acid
(BCA)) (
see
Note 3
).
10. Spectrophotometer.
11. Benchtop centrifuge.
1. Precast Tris-Glycine (Laemmli) SDS-PAGE gel with a uniform,
12 % acrylamide separation gel (
see
Note 4
). Recommended
dimensions: 18 cm × 16 cm × 1 mm, recommended comb: 10
wells, 1 mm thickness.
2. Vertical slab gel electrophoresis apparatus (e.g., Hoefer SE600).
3. 1-200 μl Gel-saver loading tip or Hamilton syringe.
4. 1× Tris-Glycine (Laemmli) SDS-PAGE running buffer.
5. Molecular weight marker.
6. Power supply.
7. Coomassie brilliant blue staining solution: 1 g Coomassie bril-
liant blue R250, 0.25 g Coomassie brilliant blue G250, 210 ml
ethanol, 25 ml methanol, 50 ml acetic acid, 215 ml H
2
O.
8. Coomassie destaining solution: 45 % (v/v) methanol, 10 %
(v/v) acetic acid, 45 % (v/v) H
2
O.
2.2
SDS-PAGE
2.3
Cutting the Gel
1. Glass plate.
2. Scalpel.
3. Eppendorf Safe Lock tubes.
1. Ammonium hydrogen carbonate buffer: 100 mM NH
4
HCO
3
(adjust pH 8.5 with NH
4
OH).
2. Destaining solution: Bring 30 ml acetonitrile (ACN) to a final
volume of 100 ml with ammonium hydrogen carbonate buffer.
2.4 In-Gel Digest
(See Note 5)
