Improved Native Isolation of Endogenous Protein A-Tagged Protein Complexes | Zendy

John LaCava | Zendy; Nagarajan Chandramouli | Zendy; Hongbing Jiang | Zendy; Michael P. Rout | Zendy

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Improved Native Isolation of Endogenous Protein A-Tagged Protein Complexes

Author(s) -

John LaCava,

Nagarajan Chandramouli,

Hongbing Jiang,

Michael P. Rout

Publication year - 2013

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/000114012

Subject(s) - reagent , elution , chromatography , chemistry , protein purification , size exclusion chromatography , trimer , peptide , affinity chromatography , protease , biochemistry , dimer , enzyme , organic chemistry

Here we report a modified peptide reagent useful for the rapid, native elution of protein complexes containing a Protein-A-tagged component. We tested this reagent for the elution of tagged endogenous protein complexes from yeast (Nup53p/Nup170p dimer; Nup1p/Kap95p/Kap60p trimer; pentameric GINS complex) and bacteria (RNAP holoenzyme). The majority of the affinity-isolated material is released within 15 minutes under mild conditions, and the elution reagent itself is readily depleted from the elution mixture by simple spin column gel filtration. This reagent is ideal for eluting protein complexes after Protein A / IgG affinity isolation when protease cleavage is not possible or not desirable and facile depletion of the elution reagent is needed.

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