Extraction of cytokeratin from the human submandibular gland and its electrophoretic analysis

We evaluated a method for extracting cytokeratin (CK) from the normal human submandibular gland and analyzed CK distribution by one- and two-dimensional electrophoresis. Four submandibular glands without histological changes under a light microscope were used as specimens. Intermediate filamentous protein was extracted by a prior modified method CK was not adequately extracted in 1-2% sodium dodecyl sulfate -5% 2-mercaptoethanol solution. On the other hand, 11 bands with molecular weights of 40-58 K were obtained after extraction in 9.5 M urea -5% 2-mercaptoethanol solution. Although fragmentation of cytoskeletal protein was observed, there were no differences in bands according to the strength of homogenization. This fragmentation seemed to be due to restriction degradation by protease. Immunoblotting revealed the reaction of this CK extract to 2 pan-CK antibodies, i.e., K8.13, which recognizes type II basic CK, and C-11, which recognizes CK4, CK5, CK6, CK8, CK10, CK13, CK18. Among monospecific antibodies, anti-CK7 (LDS-68), CK8 (M20), CK13 (KS-1A3), CK18 (CY-90), and CK19 (BA17) antibodies showed positive reactions. Glial fibrillary acidic protein, neurofilament (NF) 68 K, NF160 K, or NF200 K showed no reactions. These results of one- and two-dimensional electrophoretic analysis suggest the presence of CK5, CK7, CK8, CK13, CK14, CK15, CK17, CK18, and CK19 in the normal submandibular gland.