Biology Reference
In-Depth Information
4. Notes
1.
Figure
3
shows an experimental design with the inclusion of an
internal standard. This internal standard is composed of all
proteins from both the membrane and cytosolic fractions from
the same number of cells. Such an experiment can be analyzed
using DIGE-specifi c software, such as DeCyder (GE Healthcare),
enabling meaningful information such as the standardized
abundance values to be calculated and a series of statistical tests
to be performed.
2.
Only use BD Falcon tubes as the material from which it is
constructed (polypropylene) is compatible with precipitation
agent. Polystyrene tubes are not compatible with this reagent.
3.
Resuspend the lyophilized 25 nmol DIGE fl uor minimal Cy2,
Cy3, and Cy5 CyDyes in 125 mL of anhydrous DMF to pro-
duce 200 pmol/mL stock solutions. Aliquot the stocks into
clearly labeled screw-top microfuge tubes (1 mL), which should
be fl ushed with argon before capping. The aliquots can be
stored at −80°C for >12 months without appreciable loss of
sensitivity. When required, simply add the sample directly to
the tube of dye.
4.
Keep the coversheet that is used to protect the precast gels
prior to electrophoresis. It is useful when it comes to storing
the gels in the freezer.
References
1. Mayrhofer C, Krieger S, Allmaier G, Kerjaschki
D (2006) DIGE compatible labeling of surface
proteins on vital cells
in vitro
and
in vivo
.
Proteomics 6:579-585.
2. Strober W (2001) Monitoring cell growth. In:
Coligan JE, Bierer BE, Margulies DH, Sherach
EM, Strober W (eds). Current Protocols in
Immunology, 5th edn. Wiley, New York.
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