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The study of protein recruitment to laser-induced DNA lesions can be distorted by photoconversion of the DNA binding dye Hoechst
A commonly used approach for assessing DNA repair factor recruitment in mammalian cells is to induce DNA damage with a laser in the UV or near UV range and follow the local increase of GFP-tagged proteins at the site of damage. Often these measurements are performed in the presence of the blue DNA d...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
F1000 Research Limited
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392149/ https://www.ncbi.nlm.nih.gov/pubmed/30828443 http://dx.doi.org/10.12688/f1000research.17865.2 |
Sumario: | A commonly used approach for assessing DNA repair factor recruitment in mammalian cells is to induce DNA damage with a laser in the UV or near UV range and follow the local increase of GFP-tagged proteins at the site of damage. Often these measurements are performed in the presence of the blue DNA dye Hoechst, which is used as a photosensitizer. However, a light-induced switch of Hoechst from a blue-light to a green-light emitter will give a false positive signal at the site of damage. Thus, photoconversion signals must be subtracted from the overall green-light emission to determine true recruitment. Here we demonstrate the photoconversion effect and suggest control experiments to exclude false-positive results. |
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