Generation of an alpaca‐derived nanobody recognizing γ‐H2AX
Post‐translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single‐chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we ge...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2015-01-01
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| Series: | FEBS Open Bio |
| Subjects: | |
| Online Access: | https://doi.org/10.1016/j.fob.2015.09.005 |
| _version_ | 1811299569247453184 |
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| author | Malini Rajan Oliver Mortusewicz Ulrich Rothbauer Florian D. Hastert Katrin Schmidthals Alexander Rapp Heinrich Leonhardt M. Cristina Cardoso |
| author_facet | Malini Rajan Oliver Mortusewicz Ulrich Rothbauer Florian D. Hastert Katrin Schmidthals Alexander Rapp Heinrich Leonhardt M. Cristina Cardoso |
| author_sort | Malini Rajan |
| collection | DOAJ |
| description | Post‐translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single‐chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ‐H2AX.In vitro andin vivo characterization showed the specificity of the γ‐H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers. |
| first_indexed | 2024-04-13T06:37:48Z |
| format | Article |
| id | doaj.art-ba209d7079364f15884bd6637543f620 |
| institution | Directory Open Access Journal |
| issn | 2211-5463 |
| language | English |
| last_indexed | 2024-04-13T06:37:48Z |
| publishDate | 2015-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | FEBS Open Bio |
| spelling | doaj.art-ba209d7079364f15884bd6637543f6202022-12-22T02:57:51ZengWileyFEBS Open Bio2211-54632015-01-015177978810.1016/j.fob.2015.09.005Generation of an alpaca‐derived nanobody recognizing γ‐H2AXMalini Rajan0Oliver Mortusewicz1Ulrich Rothbauer2Florian D. Hastert3Katrin Schmidthals4Alexander Rapp5Heinrich Leonhardt6M. Cristina Cardoso7Department of Biology, Technische Universitaet Darmstadt, GermanyBiozentrum, Department of Biology II, Ludwig Maximilians Universitaet Munich, GermanyPharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, GermanyDepartment of Biology, Technische Universitaet Darmstadt, GermanyChromotek GmbH, Munich, GermanyDepartment of Biology, Technische Universitaet Darmstadt, GermanyBiozentrum, Department of Biology II, Ludwig Maximilians Universitaet Munich, GermanyDepartment of Biology, Technische Universitaet Darmstadt, GermanyPost‐translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single‐chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ‐H2AX.In vitro andin vivo characterization showed the specificity of the γ‐H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.https://doi.org/10.1016/j.fob.2015.09.005ChromobodiesDNA repairPost-translational modificationsLive cell microscopyAlpaca heavy chain antibodiesLaser microirradiation |
| spellingShingle | Malini Rajan Oliver Mortusewicz Ulrich Rothbauer Florian D. Hastert Katrin Schmidthals Alexander Rapp Heinrich Leonhardt M. Cristina Cardoso Generation of an alpaca‐derived nanobody recognizing γ‐H2AX FEBS Open Bio Chromobodies DNA repair Post-translational modifications Live cell microscopy Alpaca heavy chain antibodies Laser microirradiation |
| title | Generation of an alpaca‐derived nanobody recognizing γ‐H2AX |
| title_full | Generation of an alpaca‐derived nanobody recognizing γ‐H2AX |
| title_fullStr | Generation of an alpaca‐derived nanobody recognizing γ‐H2AX |
| title_full_unstemmed | Generation of an alpaca‐derived nanobody recognizing γ‐H2AX |
| title_short | Generation of an alpaca‐derived nanobody recognizing γ‐H2AX |
| title_sort | generation of an alpaca derived nanobody recognizing γ h2ax |
| topic | Chromobodies DNA repair Post-translational modifications Live cell microscopy Alpaca heavy chain antibodies Laser microirradiation |
| url | https://doi.org/10.1016/j.fob.2015.09.005 |
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