Immunoprecipitation (IP) of A431 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.

A431 cells are a model cell line (epidermoid carcinoma) used in biomedical research. More specifically, they are used in studies of the cell cycle and cancer-associated cell signal pathways, because they express abnormally high levels of the Epidermal growth factor receptor (EGFR). As such they are often used as a positive control for EGFR expression. They contain no functional p53, a potent tumor suppressor gene,So they are highly sensitive to mitogenic stimuli. A431 cell line was established from an epidermoid carcinoma in the skin/epidermis of an 85- year-old female patient. 

Epidermal growth factor (EGF) stimulation of A431 cells induces rapid tyrosine phosphorylation of intracellular signalling proteins, which control cellular processes such as growth, proliferation and apoptosis. At low (picomolar) concentrations EGF promotes cell growth of A431 cells, while at higher (nanomolar) concentrations, it inhibits growth by causing the cells to terminally differentiate. Treatment of A431 cells with bradykinin reduces basal and EGF-induced EGFR phosphorylation. Treatment with sertoli cell secreted growth factor (SCSGF) strongly induces cell proliferation. Stimulation of A431 cells with phorbol esters induces expression of interleukin 1-related protein IL1H.