Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4153

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Tandem Affinity Protein (TAP) Purification from Yeast

David C. Amberg, Daniel J. Burke, and Jeffrey N. Strathern

This protocol was adapted from "TAP Purification," Techniques and Protocols 5, in Methods in Yeast Genetics, 2005 edition, by David C. Amberg, Daniel J. Burke, and Jeffrey N. Strathern. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

The first 100 words of the full text of this article appear below.


INTRODUCTION

This procedure is for the purification of tandem affinity protein-tagged proteins and their complexes. It can be followed by characterization of the proteins by polyacrylamide gel electrophoresis and mass spectroscopy.


MATERIALS

Reagents

Appropriate yeast culture and liquid growth medium

caution Acetone

caution caution Acetone containing 0.05 N HCl

recipe Calmodulin Binding Buffer (CBB)

Calmodulin Sepharose (Amersham)

recipe CaCl2, 1 M

caution CBB, containing 0.1% (v/v) NP-40

caution CBB, containing 0.02% (v/v) NP-40

IgG Sepharose 6 Fast Flow (Amersham)

caution Liquid N2

recipe caution NP-40 Buffer, ice cold

Protease inhibitor cocktail (e.g., Sigma)

Sepharose 6B beads (Sigma)

recipe TAP Calmodulin Elution Buffer (TCEB)

caution TCA (trichloroacetic acid), 100%

TEV protease (Invitrogen)

recipe TEV C-buffer

Equipment

Centrifuge . . . [Full Text of this Article]


METHOD


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