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A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex

Academic Article
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Overview

authors

  • Honey, S.
  • Schneider, B. L.
  • Schieltz, D. M.
  • Yates III, John
  • Futcher, B.

publication date

  • February 2001

journal

  • Nucleic Acids Research  Journal

abstract

  • A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

subject areas

  • Adaptor Proteins, Signal Transducing
  • Adenosine Triphosphatases
  • Amino Acid Sequence
  • Antibodies, Monoclonal
  • Base Sequence
  • Blotting, Western
  • CDC28 Protein Kinase, S cerevisiae
  • Calmodulin
  • Calmodulin-Binding Proteins
  • Cell Cycle Proteins
  • Chromatography, Affinity
  • Cyclin B
  • Cyclin-Dependent Kinase Inhibitor Proteins
  • Fungal Proteins
  • HSP70 Heat-Shock Proteins
  • Hemagglutinins
  • Histidine
  • Macromolecular Substances
  • Mass Spectrometry
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagenesis, Insertional
  • Nickel
  • Precipitin Tests
  • Protein Binding
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
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Identity

PubMed Central ID

  • PMC29631

International Standard Serial Number (ISSN)

  • 0305-1048

Digital Object Identifier (DOI)

  • 10.1093/nar/29.4.e24

PubMed ID

  • 11160944
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Additional Document Info

start page

  • e24

volume

  • 29

issue

  • 4

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