Protocol

Two-hybrid Systems

Stage 1: Characterization of a Bait-LexA Fusion Protein

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

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INTRODUCTION

This protocol describes how to generate a plasmid construct (pBAIT) that expresses a target protein fused to the bacterial LexA protein. pBAIT is cotransformed into yeast with a lexAop-lacZ reporter plasmid carrying the bacterial lacZ gene under the control of the lexA operator. The recipient yeast strain contains a chromosomally integrated leu2 reporter gene, also under the control of the lexA operator. pBAIT is analyzed in transformants to establish whether the bait protein is expressed as a stable nuclear protein of the correct size that does not independently activate transcription of either of the lexA operator-reporter genes to a significant . . . [Full Text of this Article]

MATERIALS

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METHOD