Making Yeast Cell Extracts for Purifying TAP-tagged Protein Complexes

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INTRODUCTIONOne approach to identifying protein–protein interactions is the biochemical purification of a target protein from cells or tissues under nondenaturing conditions followed by the mass spectrometric identification of the components of the purified protein complex. This combination of highly specific protein purification strategies and mass spectrometry has proven to be a very successful approach for identifying protein–protein interactions. The tandem affinity purification (TAP) affinity tag and purification method allows efficient recovery of proteins present at low cellular concentrations under native conditions. Expressing the target protein at its natural levels avoids the assembly of overexpressed proteins into nonphysiological complexes. This protocol describes the preparation of TAP-tagged yeast cells.

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confidence: 99%

Growing and Harvesting TAP-Tagged Yeast Cells

Link¹,

Weaver²,

Farley³

2011

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IgG Affinity Capture of TAP-tagged Protein Complexes from Cell Extracts: Affinity Purification Step 1

Link¹,

Weaver²,

Farley³

2011

INTRODUCTIONOne approach to identifying protein–protein interactions is the biochemical purification of a target protein from cells or tissues under nondenaturing conditions, followed by the mass spectrometric identification of the components of the purified protein complex. The combination of highly specific protein purification strategies and mass spectrometry has proven to be a very successful approach for identifying protein–protein interactions. The tandem affinity purification (TAP) affinity tag and purification method allows efficient recovery of proteins present at low cellular concentrations under native conditions. Expressing the target protein at its natural levels avoids the assembly of overexpressed proteins into nonphysiological complexes. This protocol describes the immunoglobulin G (IgG) affinity capture of TAP-tagged complexes. This procedure is followed by calmodulin affinity purification.

Affinity Capture of TAP-tagged Protein Complexes: Affinity Purification Step 2

Link¹,

Weaver²,

Farley³

2011

INTRODUCTIONOne approach to identifying protein–protein interactions is the biochemical purification of a target protein from cells or tissues under nondenaturing conditions, followed by the mass spectrometric identification of the components of the purified protein complex. The combination of highly specific protein purification strategies and mass spectrometry has proven to be a very successful approach for identifying protein–protein interactions. The tandem affinity purification (TAP) affinity tag and purification method allows efficient recovery of proteins present at low cellular concentrations under native conditions. Expressing the target protein at its natural levels avoids the assembly of overexpressed proteins into nonphysiological complexes. This protocol describes calmodulin affinity capture of the TAP-tagged complexes after immunoglobulin G (IgG) affinity purification has been performed. These complexes can then be analyzed by methods such as high-sensitivity liquid chromatography coupled with tandem mass spectrometry and/or multidimensional protein identification technology (MudPIT) analysis.