Protocol

Identifying DNA-binding Proteins in Bacteriophage Expression Libraries

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 identify cloned cDNAs encoding proteins that bind to specific DNA sequences. The methods used are very similar to those used for immunological screening of expression libraries except that the nitrocellulose filters carrying immobilized proteins are screened with ³²P-labeled double-stranded DNA rather than with antibodies.

MATERIALS

10x Binding buffer

10x Kinase/ligase buffer

1x Binding buffer containing 0.25% (w/v) nonfat dry milk

About 10 ml of this blocking solution is needed per 82-mm filter screened or 25 ml per 138-mm filter.

1x Binding buffer containing 1 mM DTT (dithiothreitol)

This solution is used to dilute full-strength Guanidine Denaturation . . . [Full Text of this Article]