UMMS Affiliation

Department of Radiology, Division of Cell Biology and Imaging

Publication Date

2018-10-17

Document Type

Article

Disciplines

Amino Acids, Peptides, and Proteins | Cancer Biology | Cell Biology | Enzymes and Coenzymes | Immunology and Infectious Disease | Neoplasms

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.

Keywords

Immunoprecipitation, Cloning, Immunohistochemistry techniques, Hybridomas, Monoclonal antibodies, Enzyme-linked immunoassays, Immunoblotting, Cell staining

Rights and Permissions

Copyright: © 2018 Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

DOI of Published Version

10.1371/journal.pone.0205910

Source

PLoS One. 2018 Oct 17;13(10):e0205910. doi: 10.1371/journal.pone.0205910. eCollection 2018. Link to article on publisher's site

Journal/Book/Conference Title

PloS one

Related Resources

Link to Article in PubMed

PubMed ID

30332471

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.