Improved methods for separation of human cytokeratins

The cytokeratins from human bladder and esophageal epithelia were separated using chromatographic techniques. The cytokeratins were first extracted from fresh autopsy tissue using high and low salt buffers. Urea, 8.0-9.5 M, was used to solubilize the resulting cytokeratin pellet. Imidazole was found to increase the solubility of the pellet but reducing agents such as 2-mercaptoethanol were not beneficial. DEAE ion exchange chromatography produced three fractions which were analyzed by using one and two-dimensional electrophoresis. The third fraction was shown to contain the acidic cytokeratins and was further fractionated on a moderately polar reverse phase HPLC column using an acetonitrile elution gradient. 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. HPLC fractions of the acidic proteins from human esophagus revealed seven reproducible peaks. All seven peaks were shown by Western blotting to contain an epitope found on cytokeratin 13. The results suggest that the isolation and separation procedures have produced a series of peptide products which all retain a similar epitope but which vary significantly in their hydrophobic characteristics.