A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

Pleiner, Tino; Bates, Mark; Görlich, Dirk

doi:10.1083/jcb.201709115

Cited by 135 publications

(158 citation statements)

References 38 publications

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“…Previously, nanobodies were reported to bind primary antibodies from mouse and rabbit as host species for super‐resolution microscopy . Inspired by this, we introduce here the use of DNA‐conjugated bacterially derived protein A (molecular weight 46 kDa) and protein G (molecular weight 22 kDa) molecules as small binders for primary antibodies in combination with DNA‐PAINT super‐resolution microscopy (Figure ).…”

Section: Figurementioning

confidence: 99%

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

et al. 2019

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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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Section: Figurementioning

confidence: 99%

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

et al. 2019

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“…Since the preliminary results indicated that their anti-Bacilus anthracis nanobodies were stable and functional intrabodies, Anderson et al [103] expressed them fused to beta galactosidase directly in the cytoplasm of Tuner (DE3) E. coli. Shibuya et al [105] managed to coexpress in bacterial cytoplasm nanobodies fused to complementary split intein moieties for obtaining bispecific VHH constructs by in-cell transsplicing whereas nanobodies engineered with extra cysteines or ascorbate peroxidase were functionally recovered after production in NEB express F' cells [39]. Another nanobody with intrabody features has been generated to bind to the ALFA-tag and was effectively used to detect in vivo interactions and pull-down experiments [106].…”

Section: Bacterial Cytoplasmmentioning

confidence: 99%

“…Unluckily, a direct comparison between the results obtained with conventional wetlab approaches (based on random mutagenesis of the initial candidates followed by more stringent in vitro selection steps) and in silico modeling has not yet reported but it would be interesting to evaluate the necessary efforts, resources and achievements specific of the two alternatives. Considering what has been already accomplished with conventional antibodies [122], the available data suggest that a smart conventional approach can still assure results that no in silico system has provided so far [39] but algorithms become progressively more competitive. The following examples show some encouraging results of in silico protocols that follow different strategies to anticipate the potential effect of specific (multiple) single mutations.…”

Section: In Silico Optimizationmentioning

confidence: 99%

“…At the present a large array of vectors is available to produce customized VHHs in both prokaryotic and eukaryotic systems, according to the functional characteristics that they must have to serve their final application. Finally, recombinant nanobodies directed towards different IgG epitopes have been made available recently, providing a valid clonal and simple-to-functionalize alternative to the commercial polyclonal secondary antibodies [39,40].…”

Section: Introductionmentioning

confidence: 99%

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Recombinant expression of nanobodies and nanobody-derived immunoreagents

Marco

2020

Protein Expression and Purification

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A B S T R A C TAntibody fragments for which the sequence is available are suitable for straightforward engineering and expression in both eukaryotic and prokaryotic systems. When produced as fusions with convenient tags, they become reagents which pair their selective binding capacity to an orthogonal function. Several kinds of immunoreagents composed by nanobodies and either large proteins or short sequences have been designed for providing inexpensive ready-to-use biological tools. The possibility to choose among alternative expression strategies is critical because the fusion moieties might require specific conditions for correct folding or posttranslational modifications. In the case of nanobody production, the trend is towards simpler but reliable (bacterial) methods that can substitute for more cumbersome processes requiring the use of eukaryotic systems. The use of these will not disappear, but will be restricted to those cases in which the final immunoconstructs must have features that cannot be obtained in prokaryotic cells. At the same time, bacterial expression has evolved from the conventional procedure which considered exclusively the nanobody and nanobody-fusion accumulation in the periplasm. Several reports show the advantage of cytoplasmic expression, surface-display and secretion for at least some applications. Finally, there is an increasing interest to use as a model the short nanobody sequence for the development of in silico methodologies aimed at optimizing the yields, stability and affinity of recombinant antibodies.

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“…We have demonstrated that these boron probes are potential labeling probes for total and specific molecular imaging using non-optical nanoSIMS.T he probes work as small tags,w hich can be directly coupled to their targets, either by co-translational incorporation of alkyne-modified amino acids followed by click chemistry or by immunostaining with nanobodies.T his should also enable their use in amore general way,byc oupling similar probes to secondary nanobodies [30] (directed against mouse or rabbit antibodies), which should render the probes immediately applicable in any type of antibody-based imaging and in any type of sample.…”

Section: Angewandte Chemiementioning

confidence: 99%

Boron‐Containing Probes for Non‐optical High‐Resolution Imaging of Biological Samples

et al. 2019

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Boron has been employed in materials science as amarker for imaging specific structures by electron energy loss spectroscopy( EELS) or secondary ion mass spectrometry (SIMS). It has astrong potential in biological analyses as well; however,the specific coupling of asufficient number of boron atoms to abiological structure has proven challenging.Herein, we synthesizet ags containing closo-1,2-dicarbadodecaborane, coupled to soluble peptides,w hich were integrated in specific proteins by click chemistry in mammalian cells and were also coupled to nanobodies for use in immunocytochemistry experiments.T he tags were fully functional in biological samples,a sd emonstrated by nanoSIMS imaging of cell cultures.T he boron signal revealed the protein of interest, while other SIMS channelsw ere used for imaging different positive ions,such as the cellular metal ions.This allows,for the first time,the simultaneous imaging of such ions with aprotein of interest and will enable new biological applications in the SIMS field.

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A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

Abstract: Pleiner, Bates, and Görlich introduce anti–mouse and anti–rabbit IgG nanobodies that can be produced in E. coli and fused to reporters or labeled fluorescently to create bright and specific detection reagents with unique advantages over conventional polyclonal secondary antibodies.

Cited by 135 publications

References 38 publications

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

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

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Boron‐Containing Probes for Non‐optical High‐Resolution Imaging of Biological Samples

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