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Improved Native Isolation of Endogenous Protein A-Tagged Protein Complexes

LaCava, J., Chandramouli, N., Jiang, H. & Rout, M. P., Apr-2013, (Accepted/In press) In : Biotechniques. 54, 4, p. 213-216 4 p.

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    Submitted manuscript, 5 MB, PDF document

DOI

  • John LaCava
  • Nagarajan Chandramouli
  • Hua Jiang
  • Michael P. Rout

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 (RNAPII 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.

Original languageEnglish
Pages (from-to)213-216
Number of pages4
JournalBiotechniques
Volume54
Issue number4
Publication statusAccepted/In press - Apr-2013
Externally publishedYes

    Keywords

  • AFFINITY PURIFICATION, BINDING, IGG, DOMAINS, POLYMERASE, RESOLUTION, ELUTION

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