Characterization of a murine monoclonal antibody that detects a C-terminal fragment of the raf oncogene product.
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.
Giardina
S L
SL
Program Resources, Inc., National Cancer Institute-Frederick Cancer Research Facility, MD 21701.
Storm
S M
SM
Longo
D L
DL
Mathieson
B J
BJ
Rapp
U
U
Varesio
L
L
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D000918:Antibody Specificity; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D007118:Immunoassay; D007424:Intracellular Fluid / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D027384:Oncogene Proteins v-raf; D011233:Precipitin Tests; D012191:Retroviridae Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D012191:Retroviridae Proteins; D027384:Oncogene Proteins v-raf