ArticleBiophysical journal2010
Cross-validating FRAP and FCS to quantify the impact of photobleaching on in vivo binding estimates.
Article in Biophysical journal, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 52 papers.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
52 citing papers in PubMed, 91 citations in OpenAlex.
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- Single-molecule imaging for investigating the transcriptional control.Molecules and cells · 2025Review
- Experimentally Probing the Effect of Confinement Geometry on Lipid Diffusion.The journal of physical chemistry. B · 2024Article
- Combined SPT and FCS methods reveal a mechanism of RNAP II oversampling in cell nuclei.Scientific reports · 2023Article
- Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells.Sensors (Basel, Switzerland) · 2022Article
- Transcription Factor Dynamics.Cold Spring Harbor perspectives in biology · 2021Review
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- An intrinsically disordered region-mediated confinement state contributes to the dynamics and function of transcription factors.Molecular cell · 2021Article
- Concerted localization-resets precede YAP-dependent transcription.Nature communications · 2020Article
- Stoichiometric analysis of protein complexes by cell fusion and single molecule imaging.Scientific reports · 2020Article
- An alternative framework for fluorescence correlation spectroscopy.Nature communications · 2019Article
- Quantifying Single mRNA Translation Kinetics in Living Cells.Cold Spring Harbor perspectives in biology · 2018Review
- Nuclear dynamics of the Set1C subunit Spp1 prepares meiotic recombination sites for break formation.The Journal of cell biology · 2018Article
- SPT and Imaging FCS Provide Complementary Information on the Dynamics of Plasma Membrane Molecules.Biophysical journal · 2018Article
- Visualizing transcription factor dynamics in living cells.The Journal of cell biology · 2018Review
Corrections and comments
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
Abstract
Binding can now be quantified in live cells, but the accuracy of such measurements remains uncertain. To address this uncertainty, we compare fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) measurements of the binding kinetics of a transcription factor, the glucocorticoid receptor, in the nuclei of live cells. We find that the binding residence time measured by FRAP is 15 times longer than that obtained by FCS. We show that this discrepancy is not likely due to the significant differences in concentrations typically used for FRAP and FCS, nor is it likely due to spatial heterogeneity of the nucleus, improper calibration of the FCS focal volume, or the intentional FRAP photobleach. Instead, our data indicate that photobleaching of bound molecules in FCS is mainly responsible. When this effect is minimized, FRAP and FCS measurements nearly agree, although cross-validation by other approaches is now required to rule out mutual errors. Our results demonstrate the necessity of a photobleach correction for FCS measurements of GFP-tagged molecules that are bound for >0.25 s, and represent an important step forward in establishing a gold standard for in vivo binding measurements.
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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.