Synthesis and Validation of Cyanine-Based Dyes for DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 84

4. Notes

1. We have no information on the counterions of these fi nal

quaternary ammonium salt dyes, but they are almost certainly
not acetate since no peak for an acetate group appears in the
proton NMR of the fi nal compounds.
2. Charged urea/thiourea degradation products may cause protein
carbamylation, and therefore it is advisable to deionize urea and
thiourea before use. To prepare 25 mL of the deionized buffer,
add 10.5 g of urea and 3.8 g thiourea to 11 mL of milli-Q water.
Fill a small column with 7-9 g of ion-exchange resin AG501X8
(Bio-Rad) and wash it with milli-Q water. After water removal,
pass the urea/thiourea solution through the column 3-4 times
until the solution conductivity is around 0.2-0.4 mS. Add 1 g of
CHAPS, 0.33 mL of 1.5 M Tris-HCl, pH 8.8, and water to
make a 25-mL solution and then fi lter it through a 0.45-mm
fi lter. For longer-term storage, distribute the lysis buffer into
1.5-mL Eppendorf tubes and store at −80°C.

3. The identifi cations of these and other proteins affected by Fis

at the early logarithmic phase of E. coli cell growth will be pub-
lished elsewhere.

4. Dye precursors, such as (6) , (12) , and (18) (see Figs. 1 - 3 ),

could be successfully stored as dry solids at −20°C under argon,
so when more NHS dyes are needed, one last reaction could
be performed to convert the acid form of the dye into its NHS
ester.

Acknowledgments

Wan-Joong Kim and Nuraly K. Avliyakulov contributed equally to
this study. We thank the Dean's Offi ce of the David Geffen School
of Medicine at UCLA and Senior Associate Dean Leonard Rome
for the generous and continuing support of this work.

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