Analysis of Protein Posttranslational Modifications Using DIGE-Based Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 131

the study; NHS-Cy3 is used to label a separate aliquot of the same
sample, and NHS-Cy5, the IC (Fig. 1b ), with all three run on a
single gel. To minimize the effects of any day-to-day variability, at
least one sample from each experimental group is run at the same
time, for example, four gels in the same tank. Furthermore, once
complete, the oxidized and “total” protein data is analyzed sepa-
rately but using the same ICs.

2. Materials

All materials are prepared with Milli-Q water (18 ) and stored at

room temperature unless otherwise indicated. Analytical grade
reagents are used whenever possible.

Ω

1. 1.5 M Tris-HCl buffer (pH 8.5): Add approximately 100 mL

of water to a glass beaker. Weigh 36.3 g of Tris and transfer to
the beaker. Mix and adjust pH with HCl (see Note 1). Make
up to 200 mL with water and store at 4°C.

2.1. Sample
Preparation

2. 20% CHAPS: Weigh 10 g of CHAPS and transfer to a 50-mL

centrifuge tube. Make up to 50 mL with water and mix until
dissolved.

3. Lysis buffer (8 M urea, 4% CHAPS, 20 mM Tris-HCl (pH

8.5)): Add approximately 10 mL of water to a 50-mL beaker.
Weigh 12 g of urea and transfer to the beaker. Add 5 mL of
20% CHAPS and 333

L of 1.5 M Tris-HCl buffer (pH 8.5).
Mix and adjust volume to 25 mL with water. Make 1 mL ali-
quots and store at −20°C.

μ

4. Complete protease inhibitors: Dissolve one tablet of Complete

Mini, EDTA-free protease inhibitor cocktail (Roche,
Indianapolis, IN, USA) in 1 mL of water. Store at −20°C.

5. Protein Phosphatase Inhibitor Set (Millipore, Billerica, MA,

USA). This set contains inhibitors against serine/threonine
phosphatases, tyrosine phosphatases, and acid and alkaline
phosphatases. Store at −20°C.
6. Complete lysis buffer: Add 10 L of complete protease inhibitors

solution to 1 mL of lysis buffer. Add 1

μ

μ L of each Protein

Phosphatase Inhibitor per 100 μ L of lysis buffer. Store at −20°C.

7. Microcentrifuge pestles optimized for 1.5-mL tubes (USA

Scientifi c, Ocala, FL, USA).

1. Calf intestinal alkaline phosphatase (NEB) at 10,000 U/mL.

2.2. Posttranslational
Modifi cations

2. 10×Reaction buffer (NEB): 50 mM Tris-HCl, 100 mM

NaCl, 10 mM MgCl 2 , and 1 mM dithiothreitol, pH 7.9 at
25°C.

2.2.1. Phosphorylation
( see Note 2 )