Protein Protocols Protein Protocols

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utilized computational tools, applying statistical analyses to several experiments to
distill the true differences away from those induced by inter-gel fluctuations. However,
these efforts have been at best partially successful, since running different gels in order
to compare different samples has been a requirement given that all conventional protein
detection methods are nonspecific in nature. At the same time, the material and time
investment required in a technique that is already considered to be difficult has
increased.
Difference gel electrophoresis (DIGE) was developed to overcome the irreproducibility
problem in 2-DE methodology by labeling two samples with two different
fluorescent dyes prior to running them on the same gel (6). The fluorescent dyes used in
DIGE, Cy3 and Cy5 (Fig. 1), are cyanine based, molecular weight matched, amine
reactive, and positively charged. These characteristics, coupled with substoichiometric
labeling, result in no electrophoretic mobility shifts arising between the two differentially
labeled samples when coelectrophoresed. Thus, in DIGE, every identical protein
in one sample superimposes with its differentially labeled counterpart in the other
sample, allowing for more reproducible and facile detection of differences. Furthermore,
DIGE is a sensitive technique, capable of detecting a single protein between two
otherwise identical samples at levels as low as 0.01% of total protein and having an
overall detection sensitivity equal to that of silver staining.
2. Materials
All references to H2O should be read as double-distilled H2O unless stated otherwise.
In recipes, the information given in each line corresponds to final concentration, the
name of ingredient, and the amount used, in that order.
2.1. Sample Solubilization and Labeling
1. 40% Methylamine in water, urea, thiourea, dithiothreitol (DTT) and 3-[3-cholamidopropyl)
dimethyl ammonio]-1-propanesulfonate (CHAPS) are available from Sigma-Aldrich (Milwaukee,
WI). N-[2-hydroxyethyl] piperazine N'-2'-ethanesulfonic acid (HEPES) was from
Fisher Scientific (Pittsburgh, PA).
2. Lysis buffer:
8 M Urea 24.0 g
(Alternatively, 6 M urea and 2 M thiourea may be used. See Note 1)
6 M Urea 18.0 g
2 M Thiourea 7.6 g
Make up to 40 mL with HPLC quality H2O and dissolve the urea.
2% CHAPS (Sigma-Aldrich) 1.0 g
10 mM DTT 500 µL of 1 M stock or 0.077 g of solid
10 mM NaHEPES, pH 8.0 5.0 mL of 100 mM stock (Add last, see Notes 2 and 3)
Make up to 50.0 mL with HPLC quality H2O and store at –80°C in 1- to 1.5-mL aliquots.
3. Labeling solution: We typically label samples in lysis buffer with no further modification.
Note that we do not add Pharmalytes to this solution (see Note 4).
4. Quenching solution: 5 M methylamine in 100 mM NaHEPES, pH 8.0. Dissolve 2.38 g of
HEPES in 38.8 mL of 40% methylamine aqueous solution. Slowly add approx 60 mL
concentrated HCl with stirring. Cool the solution on ice and measure the pH as the HCl is
added until the pH reaches 8.0.