2D DIGE Saturation Labeling for Minute Sample Amounts - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 96

series, conditions should not be changed at all with respect to these
parameters.
Reduction of Cys-Cys bonds in proteins is essential to make
them accessible to CyDye labeling. This is performed using the
agent TCEP, not containing a sulfhydryl group, and the amount of
TCEP necessary is proportional to the amount of CyDye necessary
for optimal labeling. The molar ratio of TCEP:dye is always kept at
1:2 to ensure effi cient labeling.
The protocol developed in the following procedure is used
for all analytical labeling experiments throughout the satura-
tion DIGE experiment.
For the labeling of 5 g protein (see Note 8) in the recom-

mended lysis buffer at a concentration of 0.55-5

μ

L, typically
2 nmol TCEP and 4 nmol dye have to be added. Within this range
of protein concentration, no adjustments must be made with
respect to the reaction kinetic.
Careful optimization following the scheme in Table 1 is
strongly recommended. Since this is a laborious task comprising six
2D gels, and since changes in the sample preparation will require a
new optimization experiment, the lysis protocol should have been
successfully established before the optimization experiment. To
check for appropriate lysis conditions, label a sample following the
protocol outlined for gel number 3 in Table 1 . If necessary, opti-
mize your lysis protocol and eventually your 2D electrophoresis
conditions until the spot pattern obtained perfectly meets your
needs with respect to number of resolved protein spots visible, spot
resolution, and absence of extensive streaking.
The IPS would generally represent the individual samples best;
however, not all biological samples might already be available when

μ g/ μ

Table 1
Pipetting scheme, amounts, and fi nal concentrations for the optimization
experiment of the sample labeling

Volume ( m L)
of CyDye
solution
(2 mmol/L)

Final
concentration
of CyDye
(mmol/L)

Volume ( m L)
of TCEP solution
(2 mmol/L)

Amount
of TCEP
(nmol)

Final
concentration
of TCEP (mmol/L)

Amount
of CyDye
(nmol) Gel number

1 0.50 1.00 0.095 1.00 2.00 0.190

2 0.75 1.50 0.133 1.50 3.00 0.267

3 1.00 2.00 0.167 2.00 4.00 0.333

4 1.25 2.50 0.196 2.50 5.00 0.392

5 1.50 3.00 0.222 3.00 6.00 0.444

6 2.00 4.00 0.267 4.00 8.00 0.533