Analysis of Protein Posttranslational Modifications Using DIGE-Based Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 132

2.2.2. Oxidation

AlexaFluor 488 C 5 -aminooxyacetamide is light sensitive and very
unstable while in solution. Prepare all stock solutions on ice and
protect from light using aluminum foil or amber microcentrifuge
tubes:

1. 1 mM AlexaFluor 488 C 5 -aminooxyacetamide: Add 1.117 mL
of water to 1 mg of AlexaFluor 488 C 5 -aminooxyacetamide
(Sigma, St. Louis, MO, USA). Make 7.5 μ L aliquots in amber

L microcentrifuge tubes. Lyophilize (or dry down) while
protected from light (see Note 3). Store lyophilized aliquots at
−80°C in a light safe freezer box (cardboard).
2. 1 M ascorbic acid stock: Weigh 1.761 g of ascorbic acid and
make up to 10 mL with water. Store aliquots at −20°C.

500- μ

1. 1 M Tris-HCl buffer (pH 7.4): Add approximately 100 mL of
water to a glass beaker. Weigh 24.2 g of Tris and transfer to the
beaker. Mix and adjust to pH 7.4 with HCl (see Note 1). Make
up to 200 mL with water and store at 4°C.
2. 0.5 M MgCl 2 : Add approximately 100 mL of water to a glass
beaker. Weigh 9.52 g of MgCl 2 (anhydrous) and transfer to the
beaker. Mix and adjust volume to 200 mL.
3. 588

2.2.3. Ubiquitination

M ubiquitin aldehyde: Prepare fresh as required. Make a
1/100 dilution of the 58.8 mM stock supplied (Santa Cruz,
Santa Cruz, CA, USA). Take 1

μ

μ L of stock and transfer to a

L of water and mix.
4. 188.7 mM AMP-PNP (adenylyl-imododiphosphate, tetra-
lithium salt; Santa Cruz): Weigh 100 mg AMP-PNP into a
1.5-mL tube. Add 1 mL of water and mix. Store at −80°C.
5. 10 mM MG132 stock: Add 210

500- μ L centrifuge tube. Add 99 μ

L of DMSO per 1 mg of
MG132 (Boston Biochem, Boston, MA, USA). Mix and store
at −20°C.
6. 0.5 M ATP stock: Add 108.9

μ

L of water to a vial containing
30 mg of ATP (disodium hydrate; Sigma). Mix and store at
−20°C.
7. 0.5 M creatine phosphate stock: Add 196

μ

L of water to a vial
containing 25 mg of creatine phosphate (EMD Biochemicals,
Gibbstown, NJ, USA). Mix and store at −20°C.
8. 2× Ubiquitin conjugation buffer (100 mM Tris-HCl, pH 7.8,
10 mM MgCl 2 , 1 mM DTT, 150

μ

M ubiquitin
aldehyde, 5 mM AMP-PNP, 20 mM ATP, 20 mM creatine
phosphate, 0.5 mg/mL creatine phosphokinase): Prepare fresh
as required. Weigh 0.5 mg of creatine phosphokinase (~150 U)
and transfer to a 1.5-mL centrifuge tube. Add 100

μ M MG132, 4 μ

μ L of

1 M Tris-HCl, pH 7.4, 20 μ L of 0.5 M MgCl 2 , 15 μ L of

10 mM MG132 stock, 6.8 μ L of 588 μ M ubiquitin aldehyde,

40 μ L of 0.5 M ATP, and 40 μ L of 0.5 M creatine phosphate.