Biology Reference
In-Depth Information
intensity, etc.), and then compared by using pre-fi xed
factors (e.g., 2.0; 1.5; 1.2-fold). This gives a good fi rst
overview for comparison of just a few samples. Comparability
of gels/sample patterns is limited. Even when used in only
one gel, it is no true and reliable quantifi cation, as it does
not take into account the sometimes occurring preferential
labeling (
7
) or quenching of components in the gel.
(c) Quantitative analysis: This can only be achieved with
including an internal standard labeled with Cy2, and using
appropriate software (e.g., DeCyder, module BVA, stand-
ing for “Biological Variation Analysis”). Details of opera-
tion can be found in the manual. This type of analysis
results in lists of differentially regulated spots with low
variation within the respective group. Statistics features of
DeCyder may be used or data exported for use in other
software.
3.
Any of the previous steps (2a-c) may raise the wish to fi nd out
more about one or several of the separated spots. One way to
do this is by staining the protein pattern in the gel with a visible,
but MS-compatible dye, followed by cutting out the respective
spot/gel plug, digesting the proteins to peptides and subjecting
this mixture to mass spectrometric analysis for identifi cation
(see Note 45). We use an MS-compatible silver-staining proto-
col to visualize the protein pattern (see next step).
4.
Optional MS-compatible silver staining: Gel cassettes are
opened after the scanning, and gels are transferred to the fi xing
solution. After initial shaking for about 1 h, the gels are left at
room temperature until the next day (see Note 46). The further
incubations are performed on a laboratory shaker: 1 h in the
sensitizer solution, 3 × 20 min in water, and 30 min in silver
nitrate solution. After a 1-min wash with water, the gel is shortly
rinsed with developer and the protein pattern is developed in
two baths of developer (approximately 5 min each, depending
on the protein load and the intensity of the pattern). The stain-
ing is stopped by a glycine solution (20 min). After three 10-min
washes in water, the gel is ready for spot cutting and further
processing for MS analysis (see Notes 47-49).
1.
Besides serum, the described method is applicable to various
types of samples:
3.8. Modifi cations
of the Method
and Applications
-
Body fl uids with lower protein content than serum/plasma
may be lyophilized, followed by dissolving in DIGE labeling
buffer. For high salt-to-protein ratios desalting by gel fi ltra-
tion, dialysis or TCA precipitation is recommended (
3.8.1. Modifi cations
8
).
Cells from cell culture should be serum-free, i.e., washed
-
with isotonic solutions and then centrifuged (see Note 50).
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