Shotgun Proteomics of Plant Plasma Membrane and Microdomain Proteins Using Nano-LC-MS/MS - Plant Proteomics: Methods and Protocols - page 463

3. TGS running buffer: 0.025 M Tris, 0.188 M glycine, 0.1 %
(w/v) SDS ( see Note 8 ).
4. 30 % (w/v) acrylamide solution: Weigh 29 g of acrylamide
monomer and 1 g methylenebisacrylamide. Add to 50 mL
water in 100 mL glass beaker and stir moderately using a stir-
ring bar. After the solids have completely dissolved, add water
up to 100 mL with a graduated cylinder and store at 4 °C,
with protection from light using a light-shielding bottle or
wrapping with aluminum foil ( see Note 9 ).
5. 10 % (w/v) SDS: Add 10 g of SDS to 50 mL water and stir
moderately using a stirring bar. Add water up to 100 mL with
a graduated cylinder.
6. 10 % (w/v) ammonium persulfate: Add 1 g of ammonium
persulfate to 8 mL water and stir moderately using a stirring
bar. Add water up to 10 mL with a graduated cylinder. Store
at 4 °C for current use or at −30 °C for long-term storage.
7. N , N , N , N -Tetramethyl-ethylenediamine (TEMED) (Wako

Pure Chemical Industries, Tokyo, Japan). Store at 4 °C.

′

All of these processes must be carefully performed at a clean bench
with gloves and clean lab coat throughout to avoid contamination
by keratin, dust, and other exogenous proteinaceous materials.

2.3.2 Tryptic Digestion
Components

1. Fixation solution: Mix 50 mL of water, 40 mL of ethanol, and
10 mL of acetic acid.
2. 0.1 M ammonium bicarbonate: Weigh 3.95 g of ammonium
bicarbonate and add to 400 mL of water in a glass beaker. Stir
moderately using a stirring bar and add water up to 500 mL
with a graduated cylinder. Store at 4 °C ( see Note 10 ).
3. Acetonitrile (LC-MS grade) (Wako Pure Chemical Industries,
Tokyo, Japan). Store at 4 °C ( see Note 10 ).
4. 25 mM ammonium bicarbonate/50 % (v/v) acetonitrile:
Mix 50 mL of acetonitrile (LC-MS grade), 25 mL of 0.1 M
ammonium bicarbonate, and 25 mL of water. Store at 4 °C
( see Note 10 ).
5. Reduction buffer: Weigh 7.7 mg DTT and add to 5 mL of
0.1 M ammonium bicarbonate in a conical tube ( see Note 11 ).
6. 55 mM iodoacetamide (IAA)/0.1 M ammonium bicarbonate:
Weigh 51 mg IAA and add to 5 mL of 0.1 M ammonium
bicarbonate in a conical tube ( see Note 11 ).
7. Protease solution: Add 2 mL of 0.1 M ammonium bicarbon-
ate into a vial containing 20 g of trypsin (Sequence grade

modifi ed, Promega KK, Tokyo, Japan). Mix well and aliquot
into twenty 0.5 mL microtubes. Store at −20 °C.

μ