Biology Reference
In-Depth Information
spot quantifi cation of a sample against a pooled internal standard
to allow accurate inter-gel protein abundance comparisons.
The BVA module processes multiple gel images, performing
gel-to-gel matching of spots and enabling quantitative compari-
sons of protein abundance across multiple gels.
The Batch Processor executes both the DeCyder DIA and
BVA processes, performing fully automated spot codetection and
inter-gel matching of multiple gel images. Once the Batch Processor
has been set up with the necessary image fi les and parameters, the
gels are processed sequentially without user intervention. The
Batch Processor module signifi cantly reduces the hands-on analysis
time required.
2. Materials
Both cell conditions, Ramos-ev and Ramos-DN cells, were pro-
vided by Dr. A. Bernad (CNIC, Madrid).
1. ProteoExtract™, subcellular proteome extraction kit
(Calbiochem; see Note 1).
2.1. Subcellular
Fractionation
2.2. Chloroform/
Methanol Precipitation
1. Chloroform, ultrapure grade.
2. Methanol, ultrapure grade.
3. Deionized H
2
O, from a Milli-Q water purifi cation system or
equivalent.
1. Lysis buffer: 30 mM Tris-HCl, pH 8.5, 7 M urea, 2 M thio-
urea, and 4% (w/v) CHAPS. Store in aliquots at −20°C.
2. pH Test strips 4.5-10.0.
3. RC DC protein assay kit (Bio-Rad): RC reagent I and RC
reagent II (see Note 2), DC protein assay reagent A (see
Note 3), protein assay reagent B (see Note 4), and protein
assay reagent S (see Note 5).
2.3. Sample
Solubilization
and Quantifi cation
of Proteins
1. Anhydrous dimethylformamide (DMF), 99.8% (DMF) (Sigma)
(see Note 6).
2. 1-mL syringe and 25G × 5/8² (0.5 × 16 mm) needle (see
Note 7).
3. CyDye DIGE fl uor Cy3 (minimal dye) (GE Healthcare).
4. CyDye DIGE fl uor Cy5 (minimal dye) (GE Healthcare).
5. CyDye DIGE fl uor Cy2 (minimal dye) (GE Healthcare).
6. 10 mM lysine (Sigma). Store in aliquots at −20°C.
2.4. CyDye DIGE Fluor
Minimal Dyes
Solutions for Labeling
Protein Samples
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