Shotgun Proteomics of Plant Plasma Membrane and Microdomain Proteins Using Nano-LC-MS/MS - Plant Proteomics: Methods and Protocols - page 462

2. Microsome (MS)-suspension medium: 10 mM KH 2 PO 4 /
K 2 HPO 4 (K-P) buffer (pH 7.8), 0.25 M sucrose. Store at 4 °C
( see Note 2 ).
3. NaCl medium: Add 1.17 g of NaCl to 180 mL MS-suspension
medium and stir moderately using a stirring bar. Add
MS-suspension medium up to 200 mL with a graduated cylin-
der. Store at 4 °C ( see Note 3 ).
4. Plasma membrane (PM)-suspension medium: 10 mM Mops-
KOH (pH 7.3), 2 mM EGTA (pH 8.0), 0.25 M sucrose.
Store at 4 °C ( see Note 4 ).
5. Two-phase partition medium: Weigh 1.45 g of polyethylene
glycol 3350 and 1.45 g dextran in a 40 mL centrifuge tube.
Add 9.3 mL MS-suspension medium and 7.3 mL NaCl
medium to the centrifuge tube and mix well by shaking.
Incubate at 4 °C overnight to completely dissolve the poly-
mers ( see Note 5 ). Prepare three tubes per sample.
6. BioRad Protein Assay Kit (BioRad Laboratories, CA, USA):
Store at 4 °C.

Ultracentrifuge rotors should be prechilled at 4 °C. 2.2 Detergent-

Resistant Membrane
Extraction
Components

1. TED buffer: 50 mM Tris-HCl (pH 7.4), 3 mM EGTA (pH
8.0), 1 mM DTT. This buffer should be freshly prepared.
2. 10 % (w/v) Triton X-100 buffer: Add 1 g of Triton X-100 to
TED buffer and then adjust to 10 mL volume. Shake the
Triton X-100 buffer with a shaker for 3 h to completely dis-
solve Triton X-100. This buffer should be freshly prepared.
3. 65, 48, 35, 30, and 5 % (w/w) sucrose solution (in TED buf-
fer): Weigh 65, 24, 17.5, 15, and 2.5 g of sucrose and dissolve
in 35, 26, 32.5, 35, and 47.5 g of TED solution, respectively.
These solutions should be freshly prepared.

2.3 In-Gel Tryptic
Digestion

If more contaminant-free samples are to be analyzed, precast gels
(PAGEL NPU-10L; ATTO Corporation, Tokyo, Japan) should be
used instead of making hand-cast gels from the following
components.

2.3.1 SDS
Polyacrylamide Gel
Components 1. Running gel solution: 1.5 M Tris-HCl, pH 8.8. Add approxi-

mately 900 mL water to a 1 L glass beaker. Add 181.7 g Tris
and stir moderately using a stirring bar. After Tris is completely
dissolved, adjust pH with HCl using a pH meter. Add water
up to 1 L with a graduated cylinder ( see Note 6 ).
2. SDS sample buffer (2×): 2 % (w/v) SDS, 50 mM Tris-HCl
(pH 6.8), 6 % (v/v) -mercaptoethanol, 10 % (w/v) glycerol,

a spatula of bromophenol blue (BPB). Store at 4 °C for cur-
rent use or at −30 °C for long-term storage ( see Note 7 ).

β