Abstract
The tandem affinity purification (TAP) tag technique has been used with success to identify under nondenaturing conditions protein complexes in yeast. The technique can be used in mammalian cells, but we found that the original technique does not yield enough recovery for the identification of proteins when mammalian cells growing in monolayer have to be used. We present here a modified TAP tag technique that allows sufficient recovery of proteins from mouse fibroblasts growing in monolayer cultures. The recovery allows protein identification by mass spectrometry.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Affinity Labels / chemistry*
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Animals
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Biotinylation
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Cell Line
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Cell Proliferation
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Embryo, Mammalian / cytology
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Escherichia coli Proteins / genetics
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Fibroblasts / chemistry
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Fibroblasts / cytology
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Humans
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Immunoprecipitation
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Insulin Receptor Substrate Proteins
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Insulin-Like Growth Factor I / pharmacology
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Mass Spectrometry
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Mice
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Multiprotein Complexes / chemistry
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Multiprotein Complexes / genetics
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Multiprotein Complexes / isolation & purification*
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Phosphoproteins / chemistry
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Phosphoproteins / genetics
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Phosphoproteins / isolation & purification*
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Plasmids
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification
Substances
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Affinity Labels
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Escherichia coli Proteins
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IRS1 protein, human
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Insulin Receptor Substrate Proteins
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Irs1 protein, mouse
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Multiprotein Complexes
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Phosphoproteins
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Recombinant Fusion Proteins
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Insulin-Like Growth Factor I