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Tandem affinity purification of functional TAP-tagged proteins from human cells.

ABSTRACT: Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.

SUBMITTER: Gregan J 

PROVIDER: S-EPMC2957861 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Tandem affinity purification of functional TAP-tagged proteins from human cells.

Gregan Juraj J   Riedel Christian G CG   Petronczki Mark M   Cipak Lubos L   Rumpf Cornelia C   Poser Ina I   Buchholz Frank F   Mechtler Karl K   Nasmyth Kim K  

Nature protocols 20070101 5

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as w  ...[more]

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