期刊
CHEMBIOCHEM
卷 20, 期 8, 页码 1032-1038出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201800743
关键词
DNA-PAINT; nanotechnology; protein binders; single-molecule studies; super-resolution microscopy
资金
- DFG [DFG JU 2957/1-1]
- ERC [680241]
- Max Planck Society
- Max Planck Foundation
- Center for Nanoscience (CeNS)
- European Union [796606]
- DFG through the Graduate School of Quantitative Biosciences Munich (QBM)
- ProjectA11 [SFB1032]
- European Research Council (ERC) [680241] Funding Source: European Research Council (ERC)
- Marie Curie Actions (MSCA) [796606] Funding Source: Marie Curie Actions (MSCA)
Current optical super-resolution implementations are capable of resolving features spaced just a few nanometers apart. However, translating this spatial resolution to cellular targets is limited by the large size of traditionally employed primary and secondary antibody reagents. Recent advancements in small and efficient protein binders for super-resolution microscopy, such as nanobodies or aptamers, provide an exciting avenue for the future; however, their widespread availability is still limited. To address this issue, here we report the combination of bacterial-derived binders commonly used in antibody purification with DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy. The small sizes of these protein binders, relative to secondary antibodies, make them an attractive labeling alternative for emerging superresolution techniques. We present here a labeling protocol for DNA conjugation of bacterially derived proteins A and G for DNA-PAINT, having assayed their intracellular performance by targeting primary antibodies against tubulin, TOM20, and the epidermal growth factor receptor (EGFR) and quantified the increases in obtainable resolution.
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