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Merck
CN
  • 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
摘要

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).

材料
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产品描述

Roche
不含EDTA的cOmplete蛋白酶抑制剂混合物, Tablets provided in glass vials
Millipore
Benzonase®核酸酶,纯度>99%, Effective viscosity reduction and removal of nucleic acids from protein solutions
Sigma-Aldrich
苯甲磺酰氟, ≥98.5% (GC)
Sigma-Aldrich
乙二胺四乙酸 二钠盐 溶液, BioUltra, pH 8.0, ~0.5 M in H2O
Sigma-Aldrich
正钒酸钠, 99.98% trace metals basis