Biology Reference
In-Depth Information
3.5. Comparative
Image Analysis
1. Rotate and crop the gel images using the Ettan DIGE Imager
software to an appropriate fi eld of interest that includes the ref-
erence markers with the lower pH end of the strip on the left.
2.
Perform comparative image analysis using DeCyder 2D. Each
gel should be processed separately in the differential in-gel
analysis (DIA) module of DeCyder prior to export to the bio-
logical variation analysis (BVA) module. In DIA, spot detection
is performed based on an estimated 10,000 spots with an exclu-
sion fi lter rejecting spots with volumes <30,000. Background
subtraction and normalization are carried out automatically.
Use the reference marker detection tool to ensure that the left
and right reference markers are highlighted correctly.
3.
Import the DIA workspaces in BVA for spot matching to a
designated master gel. Given that serum proteins contain a
large number of posttranslational modifi cations that produce
long clusters of spots on the 2D gels (see Fig. 1 ), it may be
necessary to undertake spot matching manually.
4.
Statistical analysis can be performed using the numerous tests
offered by DeCyder in both the BVA and extended data analy-
sis (EDA) modules. Alternatively, normalized volumes of
matched spots can be exported into a spreadsheet (e.g., Excel,
Microsoft, Redmond, CA, USA) using DeCyder's XML
Toolbox function. Standardized abundance values can be cal-
culated by dividing the spot volumes of the Cy3 and Cy5 chan-
nels by the Cy2 channel for each gel. The resulting values are
identical to those displayed in DeCyder. Alternative software
packages can then be used to analyze the data.
5.
Once spots of interest have been determined, specify these as
“pick” spots in the BVA experiment. Create pick lists for a
minimum of three analytical gels. Each spot of interest should
be picked from three gels to provide suffi cient material for MS
analysis.
1.
Load the generated pick lists into the Ettan spot cutting robot
software ensuring the appropriate gel is placed on the spot cut-
ting tray. Align the reference markers with the white grid lines
of the spot cutting tray to facilitate automatic recognition of
the reference markers by the software.
3.6. Proteolytic
Digestion for Protein
Identifi cation
2.
Pick the gel plugs into a 96-well plate. Multiple gels can be
successfully picked into the same plate provided that great care
is taken to ensure matched spots are dispensed into the correct
wells. It may be necessary to remove the dispensing solution
(20% ethanol) between each round of picking. Once complete,
cover the plate with a silicone sealing mat and store at 4°C
until proceeding with the tryptic digest.
3.
Remove the 20% ethanol dispensing solution from the plate
wells. Add 250 mL of 50 mM NH 4 HCO 3 to each well containing
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