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Selected Protocol from "Proteins and Proteomics: A Laboratory Manual", Richard J. Simpson.
In Vivo Isotopic Labeling of Proteins for Quantitative Proteomics
Richard J Simpson
ABSTRACT
Stable isotope coding strategies are of immense value in determining protein concentration changes in cells and
tissues triggered by regulatory stimuli (e.g., drugs and toxins) and disease (e.g., caused by mutational changes).
Recognizing and identifying the small number of proteins whose expression levels differ as a consequence of disease
or external stimuli are complicated by the complexity of biological extracts and the fact that protein
posttranslational modifications are numerous and can occur at many sites on a protein.
One way of quantifying global protein expression patterns involves in vivo labeling. Using the following protocol,
stable isotopes can be incorporated into metabolic products, and the relative difference between these products from
cells grown in normal or isotope-enriched media can be readily quantified by MS analysis. This protocol was provided
by Yoshiya Oda (Eisai Co., Ltd., Ibaraki, Japan). |
Buy this book, Z700428
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Products Available for this Protocol
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| Buffers, Solutions, and Reagents |
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| Acetone |
323772 |
Acetone |
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| Acetonitrile |
34967 |
Acetonitrile |
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| Ammonium bicarbonate |
A6141 |
Ammonium bicarbonate |
|
| Trypsin |
T6567 |
Trypsin from porcine pancreas, Proteomics Grade, Dimethylated |
|
| H2O |
W4502 |
Water, Molecular Biology Reagent |
|
| 2-Mercaptoethanol |
M6250 |
2-Mercaptoethanol, >99% |
|
| Methanol |
M1775 |
Methanol, Absolute - Acetone free |
|
| Acetic Acid |
A6283 |
Acetic acid, >99% |
|
| Potassium ferricyanide |
P8131 |
Potassium hexacyanoferrate(III), 99% |
|
| Protein extraction reagent |
Protein Extraction
Reagents |
Protein Extraction Reagents |
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| Protease inhibitor cocktail |
Protease inhibitor
cocktail |
Protein Extraction Reagents |
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| Sodium thiosulfate |
S7026 |
Sodium thiosulfate |
|
| Tris-Cl |
T5941 |
Trizma® hydrochloride, for molecular biology, Biotechnology Performance Certified, cell
culture tested |
|
| Urea |
U5378 |
Urea, for molecular biology |
|
| Triton X-100 |
T8787 |
Triton® X-100, for molecular biology |
|
| Dithiothreitol (DTT) |
D9779 |
DL-Dithiothreitol, for molecular biology |
|
| Ampholine |
Carrier Ampholytes |
Carrier Ampholytes |
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| Trichloroacetic acid (TCA) |
T8657 |
Trichloroacetic acid, for electrophoresis |
|
| SDS-PAGE sample buffer (2x sample buffer for SDS-PAGE) |
S9788 |
2× SDS Sample Buffer |
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| Glycerol |
G5516 |
Glycerol, for molecular biology |
|
| SDS |
L4390 |
Sodium dodecyl sulfate, for molecular biology |
|
| Bromophenol blue |
B0126 |
Bromophenol blue |
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| Triglycine |
G1377 |
Gly-Gly-Gly |
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| TFA |
299537 |
Trifluoroacetic acid, >99%, purified by redistillation, for protein
sequencing |
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| Cell culture growth media |
Classic Media and Salts |
Classic Media and Salts |
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