Analysis of Protein Posttranslational Modifications Using DIGE-Based Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 133

Weigh 1.54 mg of DTT into a separate 1.5-mL tube and dissolve
in 100 L to the tube containing the other

buffer components. Make up to 1 mL with water and mix.
9. 1 M HEPES buffer: Add approximately 100 mL of water to a
glass beaker. Weigh 52 g of HEPES and transfer to the beaker.
Mix and adjust to pH 7.4 with HCl (see Note 1). Make up to
200 mL with water.
10. Isopeptidase-T: Concentrations and activity of commercially
available stocks vary. Our current source (Enzo Life Sciences,
Plymouth Meeting, PA, USA) is 25

μ L of water. Add 10 μ

μ g per 12.5 μ L, with

>20 U/mg of protein.
11. UCH-L3: Concentrations and activity of commercially avail-
able stocks vary. Our current source (R&D Systems,
Minneapolis, MN, USA) is 50 μ g per 50 μ L, with >1,000 pmol/

g.
12. Deubiquitinating buffer (25 mM HEPES, pH 7.4, 10 mM
DTT, isopeptidase-T, and UCH-L3): Prepare fresh as required.
Weigh 1.54 mg of DTT and transfer to a 1.5-mL tube. Add
25

min/ μ

μ L of 1 M HEPES, 2.5 μ L of isopeptidase-T, and 5 μ L of

UCH-L3. Make up to 1 mL with water and mix.

1. 1 M N -ethylmaleimide (NEM): Prepare fresh as required and
store on ice. Weigh 125 mg of NEM into a 1.5-mL tube. Add
1 mL of ethanol and mix. Store at 4°C.
2. 1 M hydroxylamine hydrochloride: Prepare fresh as required
and store at 4°C. Weigh 69.5 mg of hydroxylamine hydrochlo-
ride and transfer to a 1.5-mL centrifuge tube. Add 1 mL of
complete lysis buffer and mix.
3. Cy-maleimide dyes: Lyophilized stocks are stored at −80°C,
protected from light. Add 50

2.2.4. Palmitoylation

L of anhydrous dimethylforma-
mide (DMF) to 1 mg of Cy-maleimide dye. Mix thoroughly.
Flush with nitrogen and protect from light. Store as 5-10

μ

μ L

aliquots at −20°C.

2.3. Total Protein
Labeling (Minimal
Labeling)

1. NHS-Cy dyes ( N -hydroxysuccinimide esters; GE Healthcare,
Pittsburgh, PA, USA) are prepared according to the manufac-
turer's instructions. Lyophilized dyes are stored at −80°C, pro-
tected from light. A 5 nmol stock is dissolved in 5 μ L of fresh

DMF, aliquoted (2, 2, 1 L), and stored at −80°C, protected

from light. Prior to use, 1.5 times the volume of fresh DMF is
added (i.e., 3, 3, 1.5

μ

L) and the aliquots are then stored at
−20°C, protected from light.
2. 10 mM lysine: Weigh 146.19 mg of lysine and transfer to a
15-mL tube. Add water up to 10 mL. Aliquot into 1.5-mL
tubes and store at −20°C.

μ