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Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells.

Bio-protocol (2017-07-12)
Michal Domanski, John LaCava
ZUSAMMENFASSUNG

The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The following protocol describes an approach to purify RNA exosome complexes from HEK-293 cells, making use of inducible ectopic expression, affinity capture, and rate-zonal centrifugation. The obtained RNA exosomes have been used successfully for proteomic, structural, and enzymatic studies (Domanski et al., 2016).

MATERIALIEN
Produktnummer
Marke
Produktbeschreibung

Sigma-Aldrich
Salzsäure, ACS reagent, 37%
Sigma-Aldrich
Monoklonaler ANTI-FLAG® M2-Antikörper in Maus hergestellte Antikörper, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
Sigma-Aldrich
Monoklonaler ANTI-FLAG® M2-Antikörper in Maus hergestellte Antikörper, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Sigma-Aldrich
Tetracyclin, 98.0-102.0% (HPLC)
Supelco
SYPRO® Rubin-Protein-Gel-Farbstoff