Biology Reference
In-Depth Information
677AA7AC1257628001D512B/$fi le/28941447AA.pdf
). Briefl y,
the software merges the gel images into one image for spot detec-
tion, and the images are then re-separated and the spot boundaries
applied to each image, followed by calculation of spot area, volume,
and peak height. Using the Batch Processor tool is an easy way to
automate the linkage of differential in-gel analysis (DIA) and
biological variation analysis (BVA) modules together. The gel con-
taining the highest number of spot features is assigned the master
gel, and manual spot matching can then be performed to correctly
match the remaining fi ve Cy2 gel images with the Cy2 master. In
DIA, spot boundaries and volumes are co-detected for Cy3, Cy5,
and Cy2 channels on each gel, and protein spot abundance is
expressed as a standard/sample ratio. In BVA, protein abundance
is compared across multiple samples using the internal standard to
normalize between gels (see Fig.
2
), and statistical analysis is per-
formed to provide average ratio and one-way ANOVA values
between samples. The DeCyder software also contains a module
called extended data analysis (EDA), which provides more detailed
examination and interpretation of results including principal
component analysis (PCA), supervised and unsupervised clustering,
and discriminant analysis.
Spots of interest determined by DeCyder-based protein quantifi ca-
tion can then be selected for LC-MS/MS protein identifi cation.
Preparative gels containing 500
3.7. Protein
Identifi cation
and Validation
g secretome sample are separated
by 2DE, and subjected to the same IEF and SDS-PAGE conditions
as the DIGE gels (see Notes 12 and 21). Following electrophoresis,
the preparative gels are fi xed in 40% aqueous methanol and 7%
aqueous acetic acid for 30 min, washed with deionized water 3
times for 10 min, and proteins are visualized by incubating the gels
in a Coomassie Blue stain such as Imperial Protein Stain (Pierce)
for 1 h. Gel spots are excised, and subjected to automated in-gel
reduction, alkylation, and tryptic digestion as described previously
(
37
). Extracted peptides are fractionated by reverse phase liquid
chromatography (LC) and analyzed by MS to reveal protein
identity (
15
).
μ
4. Notes
1.
Ensure that the medium used in washing steps is pre-warmed
to 37°C. The addition of medium should take place along the
dish wall to minimize the disruption of adherent cells. Washing
is done by gently swirling the medium around the dish several
times. This stringent washing procedure is employed to remove
the FCS present during normal culturing conditions.
Search WWH ::
Custom Search