Small-Scale Immunoprecipitation from Fission Yeast Cell Extracts.
We describe procedures for the immunoprecipitation (IP) of a molecule of interest from cell extracts under native or denaturing conditions. The methods are equally effective with antibodies that directly recognize the molecule of interest and those that recognize a generic peptide "epitope tag" that has been fused to sequences encoding the gene of interest. The diverse chemistry of intermolecular interactions and enzymatic activities means that a range of different buffer conditions must be assessed empirically to identify optimal conditions for the study of a specific target/complex in a particular assay. We describe three buffers that can serve as starting points for this empirical testing and discuss modifications that are commonly used in the optimization of assays based on immunoprecipitation.
CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX, United Kingdom Agnes.Grallert@cruk.manchester.ac.uk Iain.Hagan@cruk.manchester.ac.uk.
Agnes
A
Grallert
CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX, United Kingdom Agnes.Grallert@cruk.manchester.ac.uk Iain.Hagan@cruk.manchester.ac.uk.
Iain M
IM
Hagan
D045424:Complex Mixtures / Q000737:chemistry*; D005656:Fungal Proteins / Q000302:isolation & purification*; D047468:Immunoprecipitation / Q000379:methods*; D012568:Schizosaccharomyces / Q000737:chemistry*
D045424:Complex Mixtures; D005656:Fungal Proteins