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An improved strategy for tandem affinity purification-tagging of Schizosaccharomyces pombe genes.

ABSTRACT: Tandem affinity purification (TAP) is a method that allows rapid purification of native protein complexes. We developed an improved technique to fuse the fission yeast genes with a TAP tag. Our technique is based on tagging constructs that contain regions homologous to the target gene cloned into vectors carrying a TAP tag. We used this technique to design strategies for TAP-tagging of predicted Schizosaccharomyces pombe genes (http://mendel.imp.ac.at/Pombe_tagging/). To validate the approach, we purified the proteins, which associated with two evolutionarily conserved proteins Swi5 and Sfr1 as well as three protein kinases Ksg1, Orb6 and Sid1.

SUBMITTER: Cipak L

PROVIDER: S-EPMC2956173 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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An improved strategy for tandem affinity purification-tagging of Schizosaccharomyces pombe genes.

Cipak Lubos L Spirek Mario M Novatchkova Maria M Chen Zhiming Z Rumpf Cornelia C Lugmayr Wolfgang W Mechtler Karl K Ammerer Gustav G Csaszar Edina E Gregan Juraj J

Proteomics 20091001 20

Tandem affinity purification (TAP) is a method that allows rapid purification of native protein complexes. We developed an improved technique to fuse the fission yeast genes with a TAP tag. Our technique is based on tagging constructs that contain regions homologous to the target gene cloned into vectors carrying a TAP tag. We used this technique to design strategies for TAP-tagging of predicted Schizosaccharomyces pombe genes (http://mendel.imp.ac.at/Pombe_tagging/). To validate the approach, w ...[more]

PMID: 19750511

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