Proteomic Analysis of Redox-Dependent Changes Using Cysteine-Labeling 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 116

Modifi cations of this technique have used BODIPY FL- N -(2-
aminoethyl) maleimide and BODIPY TMR C 5 -maleimide for dual
labeling of reduced and oxidized cysteines to monitor changes in
the thiol redox state of proteins in cells cultured at different oxygen
concentrations ( 15 ). Similarly, commercial Cy3 and Cy5 maleim-
ides have been also used for differential redox labeling ( 16 ),
although the reproducibility and specifi city of maleimides for thiol
labeling has been called into question ( 17 ). It is important to note

that the ICy dyes used in the protocol here carry a net charge of +1
and would be expected to cause a shift in the p I of labeled proteins
on a 2D gel adding complexity to the analysis. This problem has
recently been circumvented with the synthesis of a pair of sulfonated
iodoacetamido-cyanine dyes which have a net charge of zero (

17 ).

2. Materials

1. Cells: human mammary luminal epithelial cells (HMLECs)

( 18, 19 ).

2.1. Cell Culture
and Hydrogen
Peroxide Treatment

2. RPMI-1640 growth medium: RPMI-1640 medium (with

25 mM HEPES and L -glutamine), supplemented with 10%
fetal bovine serum (FBS), 2 mM L -glutamine, 100 mg/mL
penicillin-streptomycin (all from Gibco/Invitrogen), 5 mg/mL
hydrocortisone and 5 mg/mL insulin.
3. Solution of 0.25% trypsin and 1 mM EDTA (Gibco/Invitrogen).
4. Hydrogen peroxide treatment: 30% hydrogen peroxide solu-

tion diluted to 0.5 mM fi nal concentration in growth media.

1. Iodoacetyl cyanine dyes 3 and 5 (ICy3 and ICy5) (see Fig. 2

and Note 1): From lyophilized powder (stored at −20°C),
reconstitute to 10 mM stock by dissolving in the appropriate
volume of anhydrous N,N -dimethylformamide (DMF). Keep
stock solutions in dark at −20°C.
2. 2D lysis buffer: 8 M urea, 4% (w/v) CHAPS, 1 mM EDTA
10 mM Tris-HCl pH 8.3. To make 100 mL, dissolve 48 g of
urea in 50 mL of distilled H 2 O. Add 4 g CHAPS, 0.5 g NP-40,
0.1 mL of 1 M EDTA, and 0.67 mL of 1.5 M Tris pH 8.8 solu-
tion. This should give a fi nal pH of 8.3. Make up to fi nal volume,
aliquot, and store at −20°C. Do not heat (see Notes 2 and 3).

2.2. Preparation of ICy
Dye-Labeled Samples
for 2D DIGE

3. DTT solution: 1.3 M DTT in H 2 O. To make 10 mL, dissolve

2 g DTT in distilled H 2 O and make to 10 mL. Aliquot and
store at −20°C. Do not heat.

4. Ampholines/Pharmalyte mix: Mix equal volumes of Ampholines

(pH 3.5-10) and Pharmalyte (pH 3-10). Store at 4°C. These
broad pH range IPG buffers can be replaced with narrow-range
buffers depending on the fi rst dimension pH range.