Biology Reference
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7. After addition of 1 mL of deionized water and replacement
of the caps, the columns were rotated end-to-end for 1 min.
Caps were removed, columns centrifuged again, and the fl ow-
through was discarded.
8. After addition of 100
L of 2D DIGE lysis buffer (or rehydrated
elution reagent; see Note 10), the columns were vortexed
several times over a period of 15 min. The columns were placed
in clean collection tubes and centrifuged for 2 min. The entire
elution step was repeated twice and the eluates were pooled.
9. For protein quantifi cation, the Cytoskeleton Advanced Protein
Assay was used according to the manufacturer's instructions
(see Note 11). Hexapeptide library-treated samples were pro-
cessed immediately or stored at −20°C for later use (long-term
storage at −80°C).
μ
The protein labeling with cyanine dyes from GE Healthcare was
performed according to the protocol provided by the manufacturer.
3.3. Protein Labeling
(Minimal Labeling
with CyDyes)
1. Briefl y, the lyophilized dyes were resolved in DMF to a stock
solution of 1 nM dye (each). These stock solutions can be
stored up to 2 months at −20°C.
2. The stock solution was diluted with DMF to a fi nal working
concentration of 400 pM (see Note 12).
3. To eliminate dye-specifi c differences, a “dye swap” was applied,
i.e., each sample was labeled crosswise with Cy3 and Cy5. In
addition, for better statistical analysis, a technical replicate per
sample was used.
4. 50
g of Proteins/gel of each sample was labeled with 400 pmol
of dye (Cy3 or Cy5). A pool of all samples (50
μ
g/gel) was
labeled with 400 pmol of Cy2 to function as internal standard
on each gel.
5. The label-reaction was accomplished for 30 min on ice and
stopped with 1
μ
L of 10 mM lysine.
6. Labeled samples were directly used for isoelectric focusing
(IEF) or stored up to 1 week at −20°C.
μ
1. IPG strips were rehydrated in rehydration buffer in a rehydra-
tion tray overnight (see Note 4).
2. Labeled samples were combined (50
3.4. Isoelectric
Focusing and Large-
Format SDS PAGE
μ
g each labeled with Cy3,
Cy5, and Cy2 per gel).
3. Prior to IEF, each labeled sample mix has to be made up to
100
L with 2D DIGE lysis buffer, DTT (fi nal concentration
of 1% (w/v)), and the IPG buffer for the selected pH range
(fi nal concentration of 1% (v/v)).
4. We achieved good IEF results by using a Multiphor II elec-
trophoresis unit and 24-cm IPG strips (pH 4-7, linear).
μ
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