A Set of Homo‐Oligomeric Standards Allows Accurate Protein Counting

Finan, Kieran; Raulf, Anika; Heilemann, Mike

doi:10.1002/anie.201505664

Cited by 46 publications

(48 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For a stoichiometric quantification of the labeling quality of the BC2-tag/bivBC2-Nb detection system, we utilized the Escherichia coli protein ferritin (FtnA) recently described as a homo-oligomeric protein standard of 24 subunits 36 . We expressed BC2-tagged, as well as SNAP-tagged FtnA-24mers in U2OS cells and performed dSTORM imaging on cell lysates immobilized on coverslips 36 .…”

Section: Resultsmentioning

confidence: 99%

A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging

Virant

Traenkle²,

Maier³

et al. 2018

Nat Commun

150

166

View full text Add to dashboard Cite

Dense fluorophore labeling without compromising the biological target is crucial for genuine super-resolution microscopy. Here we introduce a broadly applicable labeling strategy for fixed and living cells utilizing a short peptide tag-specific nanobody (BC2-tag/bivBC2-Nb). BC2-tagging of ectopically introduced or endogenous proteins does not interfere with the examined structures and bivBC2-Nb staining results in a close-grained fluorophore labeling with minimal linkage errors. This allowed us to perform high-quality dSTORM imaging of various targets in mammalian and yeast cells. We expect that this versatile strategy will render many more demanding cellular targets amenable to dSTORM imaging.

show abstract

Section: Resultsmentioning

confidence: 99%

A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging

Virant

Traenkle²,

Maier³

et al. 2018

Nat Commun

150

166

View full text Add to dashboard Cite

show abstract

“…Further, carrying out a calibration experiment using DNA origami requires extensive sample preparation including protein purification and DNA origami functionalization. Alternative calibration standards that take advantage of protein complexes with known copy numbers expressed in cells have also been developed 21,22,30,31 . The use of these calibration standards also require special care since expressing these protein complexes in cells such that they are fully assembled and matured without forming aggregates is challenging.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, sparse images of single antibodies non-specifically adsorbed to cells were used as a reference for quantifying protein copy number in super-resolution images 19,20 , however, this method does not account for the unknown labeling stoichiometry. To overcome this problem, we and others have focused on developing calibration standards that can be used in conjunction with super-resolution microscopy [21][22][23] .In particular, we recently developed a versatile approach that uses a well-defined DNA origami structure as a calibration standard for super-resolution microscopy 24 . In this approach, we functionalized the DNA-origami with a defined number of GFP proteins, labeled the GFP using antibodies and carried out super-resolution imaging.…”

mentioning

confidence: 99%

Quantifying Protein Copy Number in Super Resolution Using an Imaging-Invariant Calibration

Zanacchi

Manzo

Magrassi

et al. 2019

Biophysical Journal

View full text Add to dashboard Cite

Motor proteins are nanoscale machines that convert the energy of ATP hydrolysis into the mechanical motion of walking along cytoskeletal filaments. In doing so, they transport organelles and help maintain sub-cellular organization. We previously developed a DNA origami-based calibration approach to extract protein copy number from super-resolution images. Using this approach, we show here that the retrograde motor protein dynein is mostly present as a single motor in the cytosol, whereas a small population of dynein along the microtubule cytoskeleton forms higher-order multimers organized into nano-sized domains. We further demonstrate, using dynein as a test sample, that the DNA origami-based calibration data we previously generated can be extended to super-resolution images taken under different experimental conditions, enabling the quantification of any GFP-fused protein of interest. Our results have implications for motor coordination during intracellular trafficking as well as for using super-resolution as a quantitative method to determine protein copy number at the nanoscale level.

show abstract

“…Cell preparation. U2OS human bone osteosarcoma epithelial cells constitutively expressing Nup133-Ypet (Ypet is a YFP variant detectable by GFP nanobodies 49 ) were cultured in DMEM/F-12 (Gibco) supplemented with 10% FCS (Gibco) and 2% GlutaMAX (Gibco) at 37°C in 5% CO 2 . Cells were transferred to glass bottom dishes (P35G-0-14-C, MatTek) pretreated with 50 μg/ml fibronectin (Sigma) and 1:500 Tetraspeck fluorescent beads (T7279, Invitrogen).…”

Section: Methods Masmentioning

confidence: 99%

Automated highly multiplexed super-resolution imaging of protein nano-architecture in cells and tissues

et al. 2020

Self Cite

View full text Add to dashboard Cite

Understanding the nano-architecture of protein machines in diverse subcellular compartments remains a challenge despite rapid progress in super-resolution microscopy. While single-molecule localization microscopy techniques allow the visualization and identification of cellular structures with near-molecular resolution, multiplex-labeling of tens of target proteins within the same sample has not yet been achieved routinely. However, single sample multiplexing is essential to detect patterns that threaten to get lost in multi-sample averaging. Here, we report maS 3 TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach capable of fully automated 3D direct STORM (dSTORM) imaging and solution exchange employing a re-staining protocol to achieve highly multiplexed protein localization within individual biological samples. We demonstrate 3D super-resolution images of 15 targets in single cultured cells and 16 targets in individual neuronal tissue samples with <10 nm localization precision, allowing us to define distinct nano-architectural features of protein distribution within the presynaptic nerve terminal.

show abstract

A Set of Homo‐Oligomeric Standards Allows Accurate Protein Counting

Cited by 46 publications

References 20 publications

A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging

A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging

Quantifying Protein Copy Number in Super Resolution Using an Imaging-Invariant Calibration

Automated highly multiplexed super-resolution imaging of protein nano-architecture in cells and tissues

Contact Info

Product

Resources

About