Identification of an intermediate filament associated high molecular weight protein using monoclonal antibodies.

Monoclonal antibodies reactive to a high molecular weight protein in MDBK cells were prepared and used to localize this protein in cultured cells. An insoluble fraction of MDBK cells after extraction with Triton X-100 was used as an immunogen and an immunofluorescent cell staining method was used to screen hybridoma cells. Three mAbs recognized a protein having nearly the same mobility as that of the microtubule-associated protein 2 in SDS-PAGE but no immunological reactivity of bovine microtubule proteins to these mAbs distinguished these proteins. Immunoblotting and cell staining experiments showed its presence in the intermediate filament-nuclear matrix fraction. Double staining experiments of cells using these mAbs and antibody against vimentin cleared the association of this protein with vimentin filaments. Results of immunoelectron microscopy confirmed its localization on the intermediate filaments.