English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Site-Specifically-Labeled Antibodies for Super-Resolution Microscopy Reveal In Situ Linkage Errors

Frueh, S. M., Matti, U., Spycher, P. R., Rubini, M., Lickert, S., Schlichthaerle, T., et al. (2021). Site-Specifically-Labeled Antibodies for Super-Resolution Microscopy Reveal In Situ Linkage Errors. ACS Nano, 15(7), 12161-12170. doi:10.1021/acsnano.1c03677.

Item is

Basic

show hide
Genre: Journal Article

Files

show Files

Locators

show

Creators

show
hide
 Creators:
Frueh, Susanna M.1, Author
Matti, Ulf1, Author
Spycher, Philipp R.1, Author
Rubini, Marina1, Author
Lickert, Sebastian1, Author
Schlichthaerle, Thomas2, Author              
Jungmann, Ralf2, Author              
Vogel, Viola1, Author
Ries, Jonas1, Author
Schoen, Ingmar1, Author
Affiliations:
1external, ou_persistent22              
2Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society, ou_2149679              

Content

show
hide
Free keywords: THERMAL-STABILITY; IGG; GLYCOSYLATION; CONFORMATION; CONJUGATION; VALIDATION; GENERATIONChemistry; Science & Technology - Other Topics; Materials Science; antibodies; immunoglobulin G; transglutaminase; click chemistry; fluorescent probes; super-resolution microscopy; Monte Carlo simulations;
 Abstract: The precise spatial localization of proteins in situ by super-resolution microscopy (SRM) demands their targeted labeling. Positioning reporter molecules as close as possible to the target remains a challenge in primary cells or tissues from patients that cannot be easily genetically modified. Indirect immunolabeling introduces relatively large linkage errors, whereas site-specific and stoichiometric labeling of primary antibodies relies on elaborate chemistries. In this study, we developed a simple two-step protocol to site-specifically attach reporters such as fluorophores or DNA handles to several immunoglobulin G (IgG) antibodies from different animal species and benchmarked the performance of these conjugates for 3D STORM (stochastic optical reconstruction microscopy) and DNA-PAINT (point accumulation in nanoscale topography). Glutamine labeling was restricted to two sites per IgG and saturable by exploiting microbial transglutaminase after removal of N-linked glycans. Precision measurements of 3D microtubule labeling shell dimensions in cell lines and human platelets showed that linkage errors from primary and secondary antibodies did not add up. Monte Carlo simulations of a geometric microtubule-IgG model were in quantitative agreement with STORM results. The simulations revealed that the flexible hinge between Fab and Fc segments effectively randomized the direction of the secondary antibody, while the restricted binding orientation of the primary antibody's Fab fragment accounted for most of the systematic offset between the reporter and a-tubulin. DNA-PAINT surprisingly yielded larger linkage errors than STORM, indicating unphysiological conformations of DNA-labeled IgGs. In summary, our cost-effective protocol for generating well-characterized primary IgG conjugates offers an easy route to precise SRM measurements in arbitrary fixed samples.

Details

show
hide
Language(s): eng - English
 Dates: 2021
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: ISI: 000679406500104
DOI: 10.1021/acsnano.1c03677
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: ACS Nano
  Other : ACS Nano
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: Washington, DC : American Chemical Society
Pages: - Volume / Issue: 15 (7) Sequence Number: - Start / End Page: 12161 - 12170 Identifier: ISSN: 1936-0851
CoNE: https://pure.mpg.de/cone/journals/resource/1936-0851