Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4535

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Immunoprecipitation: Preclearing the Lysate

Ed Harlow and David Lane

This protocol was adapted from "Immunoprecipitation," Chapter 7, in Using Antibodies by Ed Harlow and David Lane. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1999.


INTRODUCTION

To reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. Rabbit serum collected from a healthy adult animal that has not been previously immunized is used because it provides a complex mixture of different antibody types. Fixed Staphylococcus aureus Cowan I (SAC), a relatively inexpensive, concentrated source of protein A is used to collect the rabbit antibodies. Precleared samples prepared according to this protocol are ready for the final immunoprecipitation step.


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