Biology Reference
In-Depth Information
3.5. Calculating
the Amount of CyDye
DIGE Fluor Minimal
Dye Required to Label
a Protein Lysate
Prior to the steps in this subheading, the experimental design
should be made in case it has not previously been designed (see
Table
1
).
It is recommended to use 400 pmol of dye for labeling 50 mg
of protein.
1.
Vortex and spin down the dye stock solution in a microfuge.
2.
In order to achieve 400 pmol/mL of working dye solution in
5 mL, take a fresh microcentrifuge tube and add 3 mL of
DMF.
3.
To the 3 mL of DMF, add 2 mL of reconstituted dye stock solu-
tion. Ensure that all dye is removed from the pipette tip by
pipetting up and down several times into the working dye
solution.
The recommended experimental design should include a
pooled internal standard from all the samples. Enough amount of
internal standard must be prepared in order to be included on
every gel in the experiment.
1.
Add a volume of protein sample equivalent to 50 mg to a
microfuge tube.
3.6. Protein Labeling
with the CyDye DIGE
Fluor Minimal Dyes
2.
Add 1 mL of working dye solution to the microfuge tube con-
taining the protein sample (50 mg of protein is labeled with
400 pmol of dye for the labeling reaction).
Table 1
Experimental design for labeling Fraction F2 (membrane and organelles) using
biological replicates from four different extractions, randomization, and inverse
labeling. Fraction F3 (nuclear) should be labeled analogously
Gel
Cy2 standard interno
Cy3
Cy5
1
50 mg (6.25 mg each/sample): WT F2
(1,2,3,4), −DN F2 (1,2,3,4)
a
50 mg WT F2 (1)
50 mg −DN F2 (2)
2
50 mg (6.25 mg each/sample): WT F2
(1,2,3,4), −DN F2 (1,2,3,4)
a
50 mg WT F2 (2)
50 mg −DN F2 (4)
3
50 mg (6.25 mg each/sample): WT F2
(1,2,3,4), −DN F2 (1,2,3,4)
a
50 mg −DN F2 (1)
50 mg WT F2 (3)
4
50 mg (6.25 mg each/sample): WT F2
(1,2,3,4), −DN F2 (1,2,3,4)
a
50 mg −DN F2 (3)
50 mg WT F2 (4)
a
WT = RAMOS-ev; dominant-negative mutant of Polm (−DN)
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