Biology Reference
In-Depth Information
that CyDye DIGE fl uors are minimal dyes labeling primary
amines. Exogenous sources of amines, such as DTT or ampho-
lytes, must not be included in the buffer prior to labeling.
3. Sonicate samples in short bursts to avoid heating. Cool on ice
between bursts.
Sonication is complete when the solution appears signifi -
cantly less cloudy than the starting solution.
4. Protein precipitation is employed to selectively separate pro-
teins in the sample from contaminating substances. Current
methods of protein precipitation (TCA precipitation, acetone
precipitation, TCA combined with acetone precipitation, etc.)
suffer from several disadvantages: incomplete precipitation,
diffi cult protein resuspension, and introduction of ions that
could interfere with IEF. Kits specifi cally designed for protein
precipitation for 2DE are commercially available (e.g., 2-D
Clean-Up Kit from GE Healthcare).
5. Electrophoretic analysis of protein samples requires accurate
quantifi cation of the sample to be analyzed. Use a standard
protein quantitation method or, alternatively, use a kit designed
specifi cally for the accurate determination of protein concen-
tration in samples to be analyzed by 2DE (e.g., PlusOne™ 2-D
Quant Kit from GE Healthcare). The recommended concen-
tration of the protein lysate required for minimal labeling is
between 5 and 10 mg/mL.
6. Store CyDyes in the dark at −20°C.
7. The quality of the DMF used in all experiment is critical to
ensure a successful protein labeling. The DMF must be anhy-
drous, and it is necessary to avoid contamination with water.
After opening, over a period of time, DMF will degrade pro-
ducing amine compounds. Amines will react with CyDyes
reducing fl uors concentration available for protein labeling.
8. Acrylamide is a neurotoxin; it is important to wear gloves and
use appropriate handling precautions.
9. Prepare the agarose sealing solution during equilibration.
10. Two kinds of experimental design can be performed: the two-
or three-color experiment. In the two-color experiment, all
samples are labeled with the same fl uor (usually Cy5) and the
internal standard with Cy3 (the internal standard is created by
pooling an aliquot of all samples and labeled with one of the
CyDye fl uors). In the second case, all samples are labeled with
two fl uors (Cy3 and Cy5), enabling dye swapping to control
any dye-specifi c effects that might result from preferential
labeling or different fl uorescence characteristics of the gel at
the different excitation wavelengths. Here, the internal standard
is labeled with Cy2. The major difference between the two
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