Volume 20, Issue 8 p. 1032-1038
Communication

Bacterially Derived Antibody Binders as Small Adapters for DNA-PAINT Microscopy

Thomas Schlichthaerle

Thomas Schlichthaerle

Faculty of Physics and Center for Nanoscience, LMU Munich, Geschwister-Scholl-Platz 1, 80539 Munich, Germany

Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

These authors contributed equally to this work.

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Dr. Mahipal Ganji

Dr. Mahipal Ganji

Faculty of Physics and Center for Nanoscience, LMU Munich, Geschwister-Scholl-Platz 1, 80539 Munich, Germany

Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

These authors contributed equally to this work.

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Alexander Auer

Alexander Auer

Faculty of Physics and Center for Nanoscience, LMU Munich, Geschwister-Scholl-Platz 1, 80539 Munich, Germany

Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

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Orsolya Kimbu Wade

Orsolya Kimbu Wade

Faculty of Physics and Center for Nanoscience, LMU Munich, Geschwister-Scholl-Platz 1, 80539 Munich, Germany

Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

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Prof. Ralf Jungmann

Corresponding Author

Prof. Ralf Jungmann

Faculty of Physics and Center for Nanoscience, LMU Munich, Geschwister-Scholl-Platz 1, 80539 Munich, Germany

Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

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First published: 27 December 2018
Citations: 20

Graphical Abstract

Single-molecule super-resolution: Bacterially derived small secondary antibody binders protein A and protein G have been employed to reduce linkage errors in super-resolution microscopy with DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT).

Abstract

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.