Immunoprecipitation: Preclearing the Lysate

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.