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Affinity Purification of Yeast Protein-interacting Metabolites for ESI-MS Analysis
Published: Apr 20, 2011 DOI: 10.21769/BioProtoc.61 Views: 15276
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Abstract
The method described here can be used to discover in vivo protein-metabolite interactions. Metabolite-protein complexes are purified from yeast cell lysates by an affinity tag that recognizes the protein of interest. The protein-bound metabolites are extracted for identification by mass spectrometry, while the protein is concurrently analyzed by gel electrophoresis. A parallel experiment using cell lysate without target protein should be used as a negative control. The metabolite extract should be analyzed within 1-2 days to avoid undesired chemical reaction.
Keywords: Protein purification
Affinity purification
Yeast
Metabolite extraction
Metabolomics
Materials and Reagents
- Cells
- 1x phosphate buffered saline (PBS)
- Methanol (mass spec grade)
- Water (mass spec grade)
- 2x laemmli buffer (for SDS-page)
- NH4Ac
- EGTA
- DTT
- PMSF
- Roche protease inhibitor tablets (Roche Diagnostics)
- Lysis buffer (see Recipes)
- Wash buffer 1 (see Recipes)
- Wash buffer 2 (see Recipes)
Equipment
- Zirconia silica beads (Bio Spec Products)
- Rabbit IgG-conjugated dynabeads
- Eppendorf protein Lobind tubes
- FastPrep cell lyser with an adapter for 2 ml tubes
- Hula mixer (Life Technologies, InvitrogenTM) or similar product
- Magnetic stand for 1.5/2.0 ml tubes
- Heat block
Procedure
Category
Systems Biology > Proteomics > Whole organism
Microbiology > Microbial proteomics > Whole organism
Biochemistry > Protein > Interaction > Protein-ligand interaction
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