1. Generation and characterization of monoclonal antibodies that recognize human and murine supervillin protein isoforms.
- Author
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Smith TC, Saul RG, Barton ER, and Luna EJ
- Subjects
- Amino Acid Sequence, Animals, Enzyme-Linked Immunosorbent Assay, Epitope Mapping, HeLa Cells, Humans, Kinetics, Membrane Proteins chemistry, Mice, Microfilament Proteins chemistry, Muscles metabolism, Protein Isoforms immunology, Rats, Antibodies, Monoclonal immunology, Membrane Proteins immunology, Microfilament Proteins immunology
- Abstract
Supervillin isoforms have been implicated in cell proliferation, actin filament-based motile processes, vesicle trafficking, and signal transduction. However, an understanding of the roles of these proteins in cancer metastasis and physiological processes has been limited by the difficulty of obtaining specific antibodies against these highly conserved membrane-associated proteins. To facilitate research into the biological functions of supervillin, monoclonal antibodies were generated against the bacterially expressed human supervillin N-terminus. Two chimeric monoclonal antibodies with rabbit Fc domains (clones 1E2/CPTC-SVIL-1; 4A8/CPTC-SVIL-2) and two mouse monoclonal antibodies (clones 5A8/CPTC-SVIL-3; 5G3/CPTC-SVIL-4) were characterized with respect to their binding sites, affinities, and for efficacy in immunoblotting, immunoprecipitation, immunofluorescence microscopy and immunohistochemical staining. Two antibodies (1E2, 5G3) recognize a sequence found only in primate supervillins, whereas the other two antibodies (4A8, 5A8) are specific for a more broadly conserved conformational epitope(s). All antibodies function in immunoblotting, immunoprecipitation and in immunofluorescence microscopy under the fixation conditions identified here. We also show that the 5A8 antibody works on immunohistological sections. These antibodies should provide useful tools for the study of mammalian supervillins., Competing Interests: We have the following interests: Research in the Antibody Characterization Program was overseen by Leidos Biomedical Research, Inc. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials. more...
- Published
- 2018
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