Biology Reference
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as “anchor points” such that the pixel coordinates from the
software can be accurately converted to picking coordinates.
3.
DMF is used to reconstitute the CyDye and should be anhy-
drous. Poor-quality DMF will result in reduced labeling effi -
ciency and reduced shelf life for the dyes. Water accumulation
and amine-containing byproducts can be avoided by the addi-
tion of a 4 Å molecular sieve (cat. no. M2635, Sigma-Aldrich ® )
to absorb these impurities and the water.
4.
Wider pH range test papers are not accurate enough.
5.
The initial determination of protein concentration should be
verifi ed using an assay that is compatible with the reagents that
are used in classical 2D gel electrophoresis. Chemicals such as
urea and DTT can interfere with standard protein assays.
Labeling should be performed at the same protein concentra-
tion across all the samples in the experiment.
6.
The incorporation of a dye swap in a 3-dye approach negates
any chance for dye bias. Utilizing a 2-dye approach will also
negate this dye bias ( 19 ).
7.
Please see the paper by Karp et al. ( 19 ) for a discussion on how
many replicates should be run in an experiment. Another paper
by Karp et al. discusses when pooling or subpooling of samples
can be employed ( 20 ).
8.
For the minimal labeling CyDyes, see also Table 2 .
Cy2 has an excitation maximum at 491 nm and emission max-
imum at 509 nm.
Cy3 has an excitation maximum at 553 nm and emission max-
imum at 569 nm.
Cy5 has an excitation maximum at 645 nm and emission max-
imum at 664 nm.
9.
The gel should only be fi xed (usually in a combination of acid
and alcohol) after the gel has been imaged since the use of
ethanol can interfere with the fl uorescent properties of the
CyDyes. The gel should not be fi xed if Western blotting will be
Table 2
The colors related to the different CyDyes used in a minimal
labeling experiment
CyDye
Reagent color
Laser excitation
Emission fl uorescence
Cy2
Yellow
Blue
Green
Cy3
Red
Green
Orange
Cy5
Blue
Red
Red
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