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  • Tandem affinity purification protocol for isolation of protein complexes from Schizosaccharomyces pombe.

Tandem affinity purification protocol for isolation of protein complexes from Schizosaccharomyces pombe.

STAR protocols (2022-02-08)
Lubos Cipak, Tomas Selicky, Jan Jurcik, Ingrid Cipakova, Michaela Osadska, Veronika Lukacova, Peter Barath, Juraj Gregan
ABSTRACT

Many cellular processes require the activities of complex molecular machines composed of several protein subunits. Insights into these systems can be gained by isolation of protein complexes followed by in vitro analyses determining the identity, posttranslational modifications, and interactions among proteins. Here, we present a protocol for tandem affinity purification (TAP) of protein complexes from the fission yeast Schizosaccharomyces pombe. The protocol employs cells expressing C-terminally TAP-tagged proteins and is suitable for the analysis of purified proteins by mass spectrometry. For complete information on the use and execution of this protocol, please refer to Cipakova et al. (2019).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium orthovanadate, 99.98% trace metals basis
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt solution, BioUltra, pH 8.0, ~0.5 M in H2O
Millipore
Benzonase® Nuclease, Purity > 99%, Effective viscosity reduction and removal of nucleic acids from protein solutions
Roche
cOmplete, EDTA-free Protease Inhibitor Cocktail, Tablets provided in glass vials