Immunoprecipitation (IP) of NIH/3T3 Cell Lysate

Immunoprecipitation / IP 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. 

NIH 3T3 cells come from a cell line established in 1962 by two scientists at the Department of Pathology in the New York University School of Medicine, George Todaro and Howard Green. They originally obtained their 3T3 cells from Swiss albino mouse embryo tissue.The 3T3 cell line has become the standard fibroblast cell line. which refers to the cell transfer and inoculation protocol for the line, and means "3-day transfer, inoculum 3 x 10^5 cells." Using this protocol, the immortal cell line begins to thrive and stabilize in cell culture after about 20-30 generations of in vitro growth. 

Swiss 3T3 cells are inhibited by temazepam and other benzodiazepines. The original cells are extremely contact inhibited, although the cell line is no longer inhibited. 3T3 cells are sensitive to sarcoma virus focus formation, as well as leukemia virus.

3T3 cells are also receptive to transformation with polyomavirus and SV40.Lysophosphatidylcholine (lyso-PC) induces AP-1 activity and c-jun N-terminal kinase activity (JNK1) by a protein kinase C-independent pathway.