Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B

Xiaojing Zheng; Yanqing Li; Junli Zhao; Dongyang Wang; Haibin Xia; Qinwen Mao

doi:10.1371/journal.pone.0152237

Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B

PLoS One. 2016 Mar 28;11(3):e0152237. doi: 10.1371/journal.pone.0152237. eCollection 2016.

Authors

Xiaojing Zheng¹, Yanqing Li¹, Junli Zhao¹, Dongyang Wang¹, Haibin Xia¹, Qinwen Mao²

Affiliations

¹ Co-Innovation Center for Qinba Regions' Sustainable Development, College of Life Sciences, Shaanxi Normal University, Xi'an, 710062, Shaanxi, P. R. China.
² Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, 60611, United States of America.

Abstract

Human FAM76B (hFAM76B) is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s) of FAM76B, murine monoclonal antibodies (MAbs) against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B) specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s) of FAM76B.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Antibodies, Monoclonal / analysis
Antibodies, Monoclonal / immunology
Antibodies, Monoclonal / metabolism*
Antibody Specificity
Antigen-Antibody Reactions
Blotting, Western
Cloning, Molecular
Enzyme-Linked Immunosorbent Assay
HEK293 Cells
Hep G2 Cells
Humans
Immunohistochemistry
Immunoprecipitation
Mice
Microscopy, Confocal
Molecular Sequence Data
NIH 3T3 Cells
Nuclear Proteins / genetics
Nuclear Proteins / immunology*
Nuclear Proteins / metabolism
Recombinant Proteins / biosynthesis
Recombinant Proteins / immunology
Recombinant Proteins / isolation & purification
Sequence Alignment

Substances

Antibodies, Monoclonal
Nuclear Proteins
Recombinant Proteins

Grants and funding

This work was supported by the Fundamental Research Funds for the Central Universities of Shaanxi Normal University (2010ZYGX015), the Fundamental Research Funds for the Central Universities (GK201301010), research grants to HX and XZ from the National Natural Science Foundation of China (No. 81272543, No. 81471772, and 81301957), a grant to HX from the Key Program of the Natural Science Foundation of Shaanxi Province, China (No. 2014JZ005) and a grant to XZ from the Natural Science Foundation of Shaanxi Province, China (No. 2014JM4113).