Proteomics & Protien Expression

Labeling of Quantitation

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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


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).

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Products Available for this Protocol
Protocol Material Description Product #  Product Name Add to Cart
Buffers, Solutions, and Reagents      
Acetone 323772 Acetone
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  
Protease inhibitor cocktail Protease inhibitor cocktail Protein Extraction Reagents  
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  
Trichloroacetic acid (TCA) T8657 Trichloroacetic acid, for electrophoresis
SDS-PAGE sample buffer (2x sample buffer for SDS-PAGE) S9788 2× SDS Sample Buffer
Glycerol G5516 Glycerol, for molecular biology
SDS L4390 Sodium dodecyl sulfate, for molecular biology
Bromophenol blue B0126 Bromophenol blue
Triglycine G1377 Gly-Gly-Gly
TFA 299537 Trifluoroacetic acid, >99%, purified by redistillation, for protein sequencing
Cell culture growth media Classic Media and Salts Classic Media and Salts  
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