title: Improved native isolation of endogenous Protein A-tagged protein complexes.
abstract: 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.
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA.
authors.forename: John
authors.initials: J
authors.lastname: LaCava
authors.forename: Nagarajan
authors.initials: N
authors.lastname: Chandramouli
authors.forename: Hua
authors.initials: H
authors.lastname: Jiang
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007202:Indicators and Reagents; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification*; D010455:Peptides / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D055503:Protein Multimerization; D013205:Staphylococcal Protein A / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000737:chemistry; D015003:Yeasts / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D046912:Multiprotein Complexes; D010455:Peptides; D013205:Staphylococcal Protein A; D011092:Polyethylene Glycols