Journal article
Evaluation of two anti-gp91 phox antibodies as immunoprobes for Nox family proteins: mAb 54.1 recognizes recombinant full-length Nox2, Nox3 and the C-terminal domains of Nox1-4 and cross-reacts with GRP 58
Biochimica et biophysica acta. Proteins and proteomics, Vol.1752(2), pp.186-196
2005
DOI: 10.1016/j.bbapap.2005.07.018
Abstract
Progress in the study of Nox protein expression has been impeded because of the paucity of immunological probes. The large subunit of human phagocyte flavocytochrome
b
558 (Cytb), gp91
phox, is also the prototype member of the recently discovered family of NADPH oxidase (Nox) proteins. In this study, we have evaluated the use of two anti-gp91
phox monoclonal antibodies, 54.1 and CL5, as immunoprobes for Nox family proteins. Sequence alignment of gp91
phox with Nox1, Nox3 and Nox4 identified regions of the Nox proteins that correspond to the gp91
phox epitopes recognized by mAb 54.1 and CL5. Antibody 54.1 produced positive immunoblots of recombinant C-terminal fragments of these homologous proteins expressed in
E. coli. Furthermore, only mAb 54.1 recognized full-length murine and human Nox3 expressed in HEK-293 cells, in immunoblots of alkali-stripped or detergent-solubilized membranes. 54.1 recognized Nox3 expression-specific proteins with Mr 30,000, 50,000, 65,000 and 88,000 for the murine protein and Mr of 38,000–58,000, 90,000, 100,000–130,000 and a broad species of higher than 160,000 for the human protein. We conclude that mAb 54.1 can serve as a probe of Nox3 and possibly other Nox proteins, if precautions are taken to remove GRP 58 and other crossreactive membrane-associated or detergent-insoluble proteins from the sample to be probed.
Details
- Title: Subtitle
- Evaluation of two anti-gp91 phox antibodies as immunoprobes for Nox family proteins: mAb 54.1 recognizes recombinant full-length Nox2, Nox3 and the C-terminal domains of Nox1-4 and cross-reacts with GRP 58
- Creators
- Danas Baniulis - Montana State UniversityYoko Nakano - University of IowaWilliam M. Nauseef - University of IowaBotond Banfi - University of IowaGuangjie Cheng - Emory UniversityDavid J. Lambeth - Emory UniversityJames B. Burritt - Montana State UniversityRoss M. Taylor - Montana State UniversityAlgirdas J. Jesaitis - Montana State University
- Resource Type
- Journal article
- Publication Details
- Biochimica et biophysica acta. Proteins and proteomics, Vol.1752(2), pp.186-196
- Publisher
- Elsevier B.V
- DOI
- 10.1016/j.bbapap.2005.07.018
- ISSN
- 1570-9639
- eISSN
- 1878-1454
- Language
- English
- Date published
- 2005
- Academic Unit
- Anatomy and Cell Biology; Otolaryngology; Internal Medicine; Infectious Diseases
- Record Identifier
- 9984284457802771
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