Biology Reference
In-Depth Information
8. Repeat step 7 two times (see Note 8), then dilute the protein
pellet of each sample in 1 mL of ice-cold 80% [v/v] acetone.
Centrifuge the samples at 17,000 ×
g
for 3 min, 4°C.
9. Discard the supernatants and dry the protein pellets under an
extractor hood at room temperature (see Note 9).
10. Finally, determine the weight of the vessels including the pellets
and subtract the value of the empty vessels for calculation of
the pellet weights (see Note 10).
The following protocol is exemplifi ed on the comparative analysis
of two related protein fractions using the CyDye™ Fluor labeling
reagents (GE Healthcare, Munich, Germany). Both fractions should
contain 100
3.2. Sample
Preparation for DIGE
g protein and should be labeled with different fl uo-
rophors. Afterwards, the samples are combined and loaded onto a
single protein gel. Additionally, a 1:1 mixture of the two protein
samples is labeled with a third CyDye fl uorophor and is used as an
internal standard to allow a comparison of relative protein spot
intensity between both individual protein samples analyzed. The
following protocol uses 2D IEF/SDS PAGE for protein separation:
μ
1. Resuspend protein samples, 100
μ
g or more each, separately in
L of CyDye labeling compatible
lysis buffer (pH 8.5) in absence of DTT. Shake the samples for
30 min at room temperature (see Note 11).
2. Centrifuge the samples at 17,000 ×
g
for 5 min, 4°C. Collect
the supernatant.
3. Labeling reaction: Transfer a volume corresponding to a protein
amount of 100
a minimum volume of 30
μ
L) of each
sample separately to a new 1.5-mL Eppendorf vessels. A minimal
volume of 10
μ
g (usually in the range of 10-20
μ
L for each protein sample is required for label-
ing reaction. For the internal reference sample (third sample),
prepare a mixture of the two samples by combining the samples
at a ratio of 1:1. Add 1
μ
L of diluted CyDye solution to each
sample. Label the reference sample with the remaining third
CyDye (e.g., Cy2). Centrifuge them briefl y and incubate for
30 min on ice in the dark (see Note 12).
4. Stop reaction: Add 10 mM lysine stock solution (one-tenth of
the volume with respect to the individual samples) to each labeling
reaction to obtain a fi nal concentration of ~1 mM lysine per
sample (e.g., add 1.1
μ
μ
L of 10 mM lysine solution to 10
μ
L
L CyDye). Incubate the samples for
10 min on ice in the dark.
5. Add to each sample the equal volume of lysis buffer containing
the double amount of DTT (i.e., 7.5 mg DTT per mL lysis
buffer, see Note 1; e.g., 12.1
sample labeled with 1
μ
μ
L of lysis buffer with ~91
μ
g
DTT to 12.1
μ
L of CyDye-labeled sample).
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