Immunoprecipitation (IP) of RAW264.7 Cell Lysate

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. It is also used to analyze protein fractions separated by other techniques such as gel filtration or density gradient sedimentation. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or intrinsically labeled cells, or in vitro-translated proteins. This unit describes a wide range of immunoprecipitation techniques, using either suspension or adherent cells lysed by various means.Usually, plasmas transfected cells or natural cells are ideal sample for immunoprecipitation.

RAW264.7

These cells are macrophage-like cell line derived from Balb/c mice. They maintain a lot of properties of macrophages including NO production, phagocytosis (beads, other), extreme sensitivity to TLR agonists and motility. They are susceptible to genetic mutation, so freezer stocks must be made from early passage number cells. They are genetically fairly heterogeneous, which can be a benefit as clones that have reduced binding to certain ligands can be isolated and propagated. They express SRA but not MARCO in nature. They may be transfected, although mortality is high and expression is not guaranteed.

This cell line is a suitable gene transfection host. The cells will pinocytose neutral red and will phagocytose latex beads and zymosan. They are capable of antibody dependent lysis of sheep erythrocytes and tumor cell targets. LPS or PPD treatment are sinobiologial for 2 days stimulates lysis of erythrocytes but not tumor cell targets.