ArticleThe Journal of biological chemistry2012
Automating single subunit counting of membrane proteins in mammalian cells.
Article in The Journal of biological chemistry, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 43 papers.
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Who cites it
43 citing papers in PubMed, 92 citations in OpenAlex.
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- A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force-Confocal Fluorescence Microscopy.ACS photonics · 2024Article
- Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy.Analytical chemistry · 2023Article
- Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells.PloS one · 2023Article
- Hetero-pentamerization determines mobility and conductance of Glycine receptor α3 splice variants.Cellular and molecular life sciences : CMLS · 2022Article
- Oligomerization state of the functional bacterial twin-arginine translocation (Tat) receptor complex.Communications biology · 2022Article
- Modeling Non-additive Effects in Neighboring Chemically Identical Fluorophores.The journal of physical chemistry. B · 2022Article
- Diffraction-Limited Molecular Cluster Quantification with Bayesian Nonparametrics.Nature computational science · 2022Article
- Systematic Assessment of the Accuracy of Subunit Counting in Biomolecular Complexes Using Automated Single-Molecule Brightness Analysis.The journal of physical chemistry letters · 2022Article
- Toward high-throughput oligomer detection and classification for early-stage aggregation of amyloidogenic protein.Frontiers in chemistry · 2022Article
- Studying KcsA Channel Clustering Using Single Channel Voltage-Clamp Fluorescence Imaging.Frontiers in physiology · 2022Article
- Optical tweezer and TIRF microscopy for single molecule manipulation of RNA/DNA nanostructures including their rubbery property and single molecule counting.Biophysics reports · 2021Article
- Point-localized, site-specific membrane potential optical recording by single fluorescent nanodiscs.Biophysical reports · 2021Article
- PySTACHIO: Python Single-molecule TrAcking stoiCHiometry Intensity and simulatiOn, a flexible, extensible, beginner-friendly and optimized program for analysis of single-molecule microscopy data.Computational and structural biotechnology journal · 2021Article
- Reply to Pisupati et al.: Evaluating single subunit counting data to find the correct stoichiometry.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- Analyzing protein dynamics from fluorescence intensity traces using unsupervised deep learning network.Communications biology · 2020Article
- Stoichiometric analysis of protein complexes by cell fusion and single molecule imaging.Scientific reports · 2020Article
- Single-molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- A Novel AβScientific reports · 2020Article
Corrections and comments
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Authors and funding
4 authors at 2 institutions in 1 country.
Funding
Abstract
Elucidating subunit stoichiometry of neurotransmitter receptors is preferably carried out in a mammalian expression system where the rules of native protein assembly are strictly obeyed. Although successful in Xenopus oocytes, single subunit counting, manually counting photobleaching steps of GFP-tagged subunits, has been hindered in mammalian cells by high background fluorescence, poor control of expression, and low GFP maturation efficiency. Here, we present a fully automated single-molecule fluorescence counting method that separates tagged proteins on the plasma membrane from background fluorescence and contaminant proteins in the cytosol or the endoplasmic reticulum and determines the protein stoichiometry. Lower GFP maturation rates observed in cells cultured at 37 °C were partly offset using a monomeric version of superfolder GFP. We were able to correctly identify the stoichiometry of GluK2 and α1 glycine receptors. Our approach permits the elucidation of stoichiometry for a wide variety of plasma membrane proteins in mammalian cells with any commercially available TIRF microscope.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.