A murine mAb, STEGI 1, was generated against a 30-kDa raf protein purified from an Escherichia coli expression vector. Immunoblot analysis confirmed that this antibody recognized the original immunizing protein as well as a 44- to 48-kDa protein from several raf-transformed cell lines. Immunoprecipitation experiments isolated a 48-kDa protein from a cell line transfected with a c-raf construct as well as from normal NIH 3T3 fibroblasts. Parallel experiments with polyvalent antiserum prepared against E. coli-derived v-raf (C terminus)-precipitated proteins with apparent Mr of 48 and 74 kDa, as had been described previously. Immunofluorescence flow cytometry of raf-transformed cell lines revealed intense intracytoplasmic staining. This staining was specifically inhibited by preincubation of STEGI 1 with purified raf 30-kDa protein. It should now be possible to more easily assess the role of the raf oncogene product in malignant transformation.

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