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Published on: April 9, 2019
Development of a new method for absolute protein quantification on 2-D gels
Peggy Baudouin-Cornu1, Gilles Lagniel, Stéphane Chédin
1CEA, iBiTecS, Service de Biologie Intégrative et Génétique Moléculaire, Laboratoire de Biologie Intégrative, Gif-sur-Yvette, France. peggy.baudouin@cea.fr
Proteomics
|September 15, 2009
Summary
This study introduces a novel method for absolute protein quantification in yeast using radioactive sulfur labeling and 2-D PAGE. The technique enables precise measurement of protein amounts per cell, advancing systems biology research.
Area of Science:
- Proteomics
- Systems Biology
- Cellular Biology
Background:
- Accurate absolute protein concentration measurements are crucial for systems biology.
- Existing methods for proteomic absolute quantification are limited.
Purpose of the Study:
- To develop a novel, precise, and reproducible method for absolute protein quantification.
- To measure and compare protein amounts per cell under different growth conditions.
Main Methods:
- Radioactive labeling of Saccharomyces cerevisiae proteins using 35S-labeled sulfur source.
- Separation of labeled proteins using 2-D polyacrylamide gel electrophoresis (PAGE).
- Quantification of individual protein radioactivity to determine cellular protein amounts.
Main Results:
- Developed a method for absolute protein quantification in yeast.
- Successfully compared protein amounts under sulfate and methionine-rich conditions.
- Demonstrated the method's simplicity, precision, and reproducibility for soluble, abundant proteins.
Conclusions:
- The proposed method offers a valuable tool for absolute protein quantification in yeast.
- This technique provides insights into protein expression changes beyond transcriptional data.
- The method is suitable for comparative proteomic studies in systems biology.
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01:22Two-dimensional Gel Electrophoresis
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The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
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01:27SDS-PAGE
Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
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