Characterization of DNA–protein complexes by capillary electrophoresis-single molecule fluorescence correlation spectroscopy

The Analyst ◽  
2001 ◽  
Vol 126 (8) ◽  
pp. 1279-1284 ◽  
Author(s):  
Dale J. LeCaptain ◽  
Matthew A. Michel ◽  
Alan Van Orden
Keyword(s):  
Capillary Electrophoresis ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Protein Complexes ◽  
Correlation Spectroscopy ◽  
Single Molecule Fluorescence ◽  
Fluorescence Correlation

scholarly journals Stoichiometric Analysis of Protein Complexes by Cell Fusion and Single Molecule Imaging

2020 ◽  
Author(s):  
Avtar Singh ◽  
Alexander L. Van Slyke ◽  
Maria Sirenko ◽  
Alexander Song ◽  
Paul J. Kammermeier ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Protein Complexes ◽  
Correlation Spectroscopy ◽  
Single Molecule Fluorescence ◽  
Single Molecule Level ◽  
Fluorescence Correlation ◽  
Cellular Environment ◽  
Low Expression

ABSTRACTThe composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level; however, these typically require artificially low expression levels to achieve the low fluorophore concentration required for single molecule imaging, or use of detergent isolation of complexes that may perturb native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this in situ dilution strategy with single molecule techniques such as in vivo Fluorescence Correlation Spectroscopy (FCS), bleach step counting for quantifying protein complex stoichiometry, and two-color single molecule colocalization, improves the quality of data obtained using these single molecule fluorescence methods. Single Protein Recovery After Dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry.SIGNIFICANCE STATEMENTQuantifying the composition and stoichiometry of protein complexes in live cells is critical to understanding mechanisms involved in their function. Here we detail a method in which protein complexes are assembled intracellularly at physiological concentrations, but then diluted to levels suitable for single-molecule fluorescence observations while still within a cellular environment. The technique permits the use of common single molecule analysis techniques such as stepwise photobleaching quantification and fluorescence correlation spectroscopy to determine stoichiometry and functional interactions while avoiding artifacts that may occur from the use of detergent isolation methods or from the artificially low expression levels sometimes used to attain single molecule observation levels.

Characterization of Pteridines: a New Approach by Fluorescence Correlation Spectroscopy and Analysis of Assay Sensitivity

Pteridines ◽  
2001 ◽  
Vol 12 (4) ◽  
pp. 147-153 ◽  
Author(s):  
U. Demel ◽  
Z. Foldes-Papp ◽  
D. Fuchs ◽  
G. P. Tilz
Keyword(s):  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Cell Mediated Immunity ◽  
Distribution Analysis ◽  
Assay Sensitivity ◽  
New Approach ◽  
Single Molecule Level ◽  
Fluorescence Correlation

Abstract In the present investigation, fluorescence con-elation spectroscopy (FCS) was used to measure the molecular motion of the pteridine derivative neopterin. However, technical limitations in the present optical setup precluded the identification of ,single neopterin molecules. FCS measurements with a fluorophore were also can-ied out for comparison. Exemplified by rhodamine green, we have introduced a concept that allows the detection, identification and analysis of assays in solution at the single-molecule level in tenns of bulk concentration. This concept is based on FCS and Poisson distribution analysis of assay sensitivity. The molecules had not to be quantified in a more concentrated fonn, or in flow and trapping experiments. The study demonstrated an ultrasensitive, reliable, rapid and direct tool for analytics and diagnostics in solution. We discuss a possible application of our new concept in activation control of cell-mediated immunity via neopterin determination.

A sensitive and microscale method for drug screening combining affinity probes and single molecule fluorescence correlation spectroscopy

The Analyst ◽  
2015 ◽  
Vol 140 (4) ◽  
pp. 1207-1214 ◽  
Author(s):  
Lingao Ruan ◽  
Di Su ◽  
Chang Shao ◽  
Jinjie Wang ◽  
Chaoqing Dong ◽  
...  
Keyword(s):  
Theoretical Model ◽  
Single Molecule ◽  
Drug Screening ◽  
Fluorescence Correlation Spectroscopy ◽  
Screening Method ◽  
Correlation Spectroscopy ◽  
Competitive Reaction ◽  
Single Molecule Fluorescence ◽  
Fluorescence Correlation ◽  
Affinity Probes

The theoretical model of drug screening method based on competitive reaction and fluorescence correlation spectroscopy.

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