The Basics of 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 14

3. Methods

This protocol describes minimal labeling. Full details for perform-
ing a saturation labeling experiment can be found in the associated
product booklet ( 16 ).

The sample is prepared as for classical 2D gel electrophoresis ( 17 ),
except that primary amines, carrier ampholytes, and thiols are
omitted from the buffers. It is then usual to concentrate the result-
ing sample (e.g., by precipitation) and resuspend it in labeling
buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 30 mM Tris-
HCl, pH 8.5) to a concentration of between 5 mg/mL and 10 mg/
mL (though 1 mg/mL to 20 mg/mL have been successfully used)
(see Note 5). The pH of this resulting sample is then checked with
pH test paper such that the pH is between 8 and 9 (and adjusted
with 50 mM NaOH if necessary). If the pH is below 8.0, then the
dye will not bind, and if the pH is over 9.0, then multiple dyes can
bind to the protein or to different amino acids.
The internal standard is prepared by pooling together equal
aliquots of all the biological replicates in the experiment (see
Table 1 ).

3.1. Sample
Preparation

The labeling protocol ( 18 ) involves the resuspension of each lyo-
philized CyDye in DMF to create a stock solution of 1 mM. To
limit any effects of photobleaching on the fl uors, all subsequent
steps are performed in the dark.
The dye-to-protein ratio is controlled at 400 pmol of dye to

3.2. Sample Labeling

50 g of protein (though 100-1,000 pmol have been successfully

used)—bulk labeling can also be performed by keeping this ratio
constant. It is recommended to label the pooled internal standard
with the Cy2 dye and then to perform a dye swap with each of the
sample types in the experiment such that an equal number are
labeled with Cy3 as with Cy5 (see Table 1 ; Notes 6 and 7). The
labeling reaction is performed on ice for 30 min, and then, the
labeling reaction is terminated by the addition of lysine to quench
any unreacted dye (for 10 min on ice).

μ

3.3. 2D Gel
Electrophoresis

The labeled samples are mixed appropriately for loading onto the
fi rst dimension IPG strips, either by in-gel rehydration, cup load-
ing, or paper-bridge loading ( 17 ). The samples are made up to the
correct volume for sample loading ensuring that the fi nal buffer
concentrations are 7 M urea, 2 M thiourea, 4% CHAPS, 0.5% car-
rier ampholyte, and 40 mM DTT.
Standard IEF separation protocols are subsequently followed
( 17 ). After the fi rst dimension a two-step equilibration procedure
is performed. This procedure saturates the IPG strip with the
SDS buffer system required for the second dimension separation.