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.
It is within the cytoplasm that most cellular activities occur, such as many metabolic pathways including glycolysis, and processes such as cell division. The concentrated inner area is called the endoplasm and the outer layer is called the cell cortex or the ectoplasm. Movement of calcium ions in and out of the cytoplasm is thought to be a signaling activity for metabolic processes.
Cytoplasm extraction buffer
10mM Hepes or Tris (pH7.5)
water to 50 mL
Resuspend pellet in 3-5mL of Cytoplasm extraction Buffer. Put into 15mL glass pestle and crush 25-30 times on ice. Spin down at 2000rpm for 5min at 4 ℃. Remove supernatant and transfer to microcentrifuge tube. Centrifuge at 14,000rpm for 15minute at 4℃. Remove supernatant. This is the cytoplasmic protein solution.