Analysis of Protein Posttranslational Modifications Using DIGE-Based Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 136

5. Clean samples (see Note 12).

6. Label aliquots of the same samples to visualize total protein as

described below in Subheading 3.3 ( 5 ).

Prepare samples as described above (see Subheading 3.1 ) in com-
plete lysis buffer containing protease and phosphatase inhibitors.
Divide the sample into two 20-

3.2.3. Ubiquitination

μ L aliquots (the amount of protein

should be around 120 g in each). Clean the sample aliquots as

previously described (see Note 12), except resuspend the pellet as
indicated below:

μ

1. To one aliquot, add 20 μ L of 2× ubiquitin conjugation buffer

and 20 L of water (see Note 14). Mix and incubate at 37°C

for 1 h with shaking.

μ

2. To the other aliquot, add 40 L of deubiquitination buffer.

Mix and incubate at room temperature for 1 h.

μ

3. Add an equal volume of complete lysis buffer to the samples

and incubate at 4°C overnight.

4. Label the samples with NHS-Cy3 or NHS-Cy5 dyes as

described below in Subheading 3.3 .

Prepare samples as described above in complete lysis buffer containing
protease and phosphatase inhibitors but do not clean the samples:
1.

3.2.4. Palmitoylation

Add 1 L of sample to give a fi nal

concentration of 10 mM NEM. Incubate overnight at 4°C
with rocking (see Note 15).

μ L of 1 M NEM per 100 μ

2. Clean the samples to remove NEM (see Note 12).

3. Resuspend the pellet in complete lysis buffer (50-100 μ L) and

divide the samples into two equal aliquots.

4. To one of the aliquots, add an equal volume of 1 M hydroxy-

lamine hydrochloride. This is the depalmitoylated sample. To
the other aliquot, add an equal volume of complete lysis buffer.
Incubate for 1 h at room temperature on a rotating platform
or vortex.

5. Clean the samples to remove the hydroxylamine hydrochloride

(see Note 12). Resuspend the pellet in 25-50 μ L of complete

lysis buffer.

6. To label the depalmitoylated sample, add 1 L of Cy-maleimide

dye and mix by vortexing. Flush the tube with nitrogen gas
and incubate the sample at room temperature for 2 h, pro-
tected from light, followed by overnight incubation at 4°C.

μ

7. Clean the sample to remove the unbound Cy-maleimide dye

(see Note 12).

8. Label the control samples, with an NHS-Cy dye of a different

wavelength than the Cy-maleimide dye, as described in
Subheading 3.3 below.