Generation of monoclonal antibodies to the zinc finger domain of the eukaryotic transcription factor Sp1.
Using a synthetic peptide that encompasses the zinc finger domain of the eukaryotic transcription factor Sp1, we produced a number of monoclonal antibodies that specifically reacted with the target antigen. Analysis by competitive inhibition assay of five of the monoclonal antibodies revealed that they all recognized a dominant epitope in the synthetic peptide and reacted strongly to recombinantly synthesized beta-galactosidase-Sp1 fusion polypeptide. To determine cellular distribution of Sp1-like molecules, cytoplasmic and nuclear proteins from human lung fibroblasts (HFL-1) and a human rhabdomyosarcoma cell line (A204) were immunoblotted and reacted with our antibodies. In addition to the well characterized 95 Kd and 105 Kd proteins, considered to be the authentic Sp1 polypeptide, a number of other cellular proteins reacted with these antibodies. Immunofluorescence staining of the cells with mAb to the zinc finger of Sp1 also revealed cell-specific differences in intracellular distribution of Sp1-like molecules. Both cytoplasmic and nuclear staining was readily observed in the rhabdomyosarcoma cells. In contrast, while some HFL-1 cells exhibited staining of only cytoplasm, both cytoplasmic and nuclear immunofluorescence was seen in others.
Katai
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Department of Medicine, University of Tennessee, Memphis.
Terato
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Raghow
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016329:Sp1 Transcription Factor / Q000032:analysis / Q000276:immunology*; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers / Q000276:immunology*
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor