دورية أكاديمية
Generation of an alpaca-derived nanobody recognizing γ-H2AX.
| العنوان: | Generation of an alpaca-derived nanobody recognizing γ-H2AX. |
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| المؤلفون: | Rajan M; Department of Biology, Technische Universitaet Darmstadt, Germany., Mortusewicz O; Biozentrum, Department of Biology II, Ludwig Maximilians Universitaet Munich, Germany., Rothbauer U; Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany., Hastert FD; Department of Biology, Technische Universitaet Darmstadt, Germany., Schmidthals K; Chromotek GmbH, Munich, Germany., Rapp A; Department of Biology, Technische Universitaet Darmstadt, Germany., Leonhardt H; Biozentrum, Department of Biology II, Ludwig Maximilians Universitaet Munich, Germany., Cardoso MC; Department of Biology, Technische Universitaet Darmstadt, Germany. |
| المصدر: | FEBS open bio [FEBS Open Bio] 2015 Sep 21; Vol. 5, pp. 779-88. Date of Electronic Publication: 2015 Sep 21 (Print Publication: 2015). |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: John Wiley & Sons Ltd Country of Publication: England NLM ID: 101580716 Publication Model: eCollection Cited Medium: Print ISSN: 2211-5463 (Print) Linking ISSN: 22115463 NLM ISO Abbreviation: FEBS Open Bio Subsets: PubMed not MEDLINE |
| أسماء مطبوعة: | Publication: Jan. 2016- : West Sussex : John Wiley & Sons Ltd. Original Publication: Amsterdam : Elsevier, 2011- |
| مستخلص: | 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 and in 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. |
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| فهرسة مساهمة: | Keywords: Alpaca heavy chain antibodies; CKM, casein kinase 2 mutant; Chromobodies; DNA repair; ELISA, enzyme linked immunosorbent assay; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; H2AX, histone H2AX; HEK293, human embryonic kidney 293 cells; KLH, keyhole limpet hemocyanin; Laser microirradiation; Live cell microscopy; MDC1, mediator of DNA damage checkpoint-1; MEF, mouse embryonic fibroblast; Post-translational modifications; RFP, red fluorescent protein; VHH, variable domain of heavy-chain antibody; XRCC1, X-ray repair cross-complementing protein 1; siRNA, short interfering RNA |
| تواريخ الأحداث: | Date Created: 20151027 Date Completed: 20151027 Latest Revision: 20181113 |
| رمز التحديث: | 20231215 |
| مُعرف محوري في PubMed: | PMC4588710 |
| DOI: | 10.1016/j.fob.2015.09.005 |
| PMID: | 26500838 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 2211-5463 |
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| DOI: | 10.1016/j.fob.2015.09.005 |