Proteomic Analysis of Redox-Dependent Changes Using Cysteine-Labeling 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 119

3. Methods

3.1. Tissue Culture

1. Culture HMLECs in 15-cm tissue culture dishes in RPMI-1640
growth media at 37°C in a 10%-CO 2 -humidifi ed incubator.
2. Split cells approximately 1:5 when confl uent. Do not over-split.

3.2. Protein
Quantifi cation

1. HMLEC cells at ~80% confl uence are washed twice with ice-
cold 0.5× PBS and then 1,000 mL of 2D-lysis buffer added per
plate. Place dishes immediately on ice.
2. Scrape cells and collect them in labeled tubes.
3. Homogenize by passage through a 25-gauge needle 10 times.
Vortex and remove insoluble material by centrifugation
(13,000 × g /10 min/4°C) and transfer supernatant to fresh
tubes.
4. Determine protein concentration using the Coomassie Protein
Assay Reagent. Make a 5 mg/mL stock of BSA in 2D-lysis
buffer and prepare serial dilutions of 0, 0.25, 0.5, 1.0, 2.5, and
5.0 mg/mL to make a standard curve. Use a 96-well fl at-bot-
tomed assay plate and make triplicate measurements for the
BSA standards and four replicates for the experimental samples.
For this, add 2 mL of sample per well and 198 mL of assay
reagent and mix without introducing bubbles. Use a plate
reader at a wavelength of 595 nm and calculate protein con-
centrations using the standard curve (see Note 5).

3.3. Cell Treatment
and Lysis

1. Add H 2 O 2 solution to growth media of HMLEC cells at ~80%
confl uence to a fi nal concentration of 0.5 mM with gentle
swirling or leave untreated. Leave treated cells for 2, 20, or
240 min.
2. Cells are washed twice with ice-cold 0.5× PBS and lysed with
1,000 mL 2D lysis buffer per plate containing ICy3/5 at
80 pmoL/mg protein (see Note 6).
3. Scrape cells and collect them in labeled tubes.
4. Homogenize by passage through a 25-gauge needle 10 times.
Vortex and remove insoluble material by centrifugation
(13,000 × g /10 min/4°C) and transfer supernatant to fresh
tubes.
5. The ICy3/5-labeled proteins are subsequently incubated on
ice in the dark for 1 h and quenched with DTT at a 65 mM
fi nal concentration.
6. Mix 150 mg ICy3- and 150 mg ICy5-labeled samples to give
300 mg total protein. Volumes are adjusted to 450 mL with
2D-lysis buffer containing 65 mM DTT and 9 mL carrier
Ampholines/Pharmalyte (1:1; v/v) (pH 3-10) and 1 mL of