ISOLATION AND SEPARATION OF HUMAN CYTOKERATINS USING VARIOUS CHROMATOGRAPHIC TECHNIQUES

Abstract

The cytokeratins from various human tissue were isolated using chromatographic techniques. The cytokeratins were first extracted from crude tissue using high and low salt buffers. It was necessary to use a denaturing agent such as urea to solubilize the resulting cytokeratin pellet. Imidazole also seemed to help solubilize the pellet and a reducing agent such as 2-Mercaptoethanol was not needed as previously believed. The acidic cytokeratins were separated from the neutral-basic cytokeratins using a DEAE ion-exchange column. The acidic cytokeratin fraction was further separated on a moderately polar reverse phase column with an acetonitrile gradient to eluted the proteins. Tetramethylammonium tetrafluoroborate was added to the mobile phase to react with any unreacted silanol groups on the stationary phase and trifluoroacetic acid was added to ion pair with the protein. The peaks were analyzed for purity using two dimensional electrophoresis and monoclonal antibodies that recognize the cytokeratins.