Self-diffusion of a highly concentrated monoclonal antibody by fluorescence correlation spectroscopy: insight into protein–protein interactions and self-association

Soft Matter ◽  
2019 ◽  
Vol 15 (33) ◽  
pp. 6660-6676 ◽  
Author(s):  
Jessica J. Hung ◽  
Wade F. Zeno ◽  
Amjad A. Chowdhury ◽  
Barton J. Dear ◽  
Kishan Ramachandran ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Protein Interactions ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Protein Protein Interactions ◽  
Fluorescence Correlation ◽  
Self Diffusion ◽  
Self Association ◽  
Insight Into

Measurement and interpretation of self-diffusion of a highly concentrated mAb with different formulations in context of viscosity and protein self-interactions.

scholarly journals Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

2015 ◽  
Vol 26 (11) ◽  
pp. 2054-2066 ◽  
Author(s):  
Yinghua Guan ◽  
Matthias Meurer ◽  
Sarada Raghavan ◽  
Aleksander Rebane ◽  
Jake R. Lindquist ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescent Protein ◽  
Stokes Shift ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Quantitative Detection ◽  
Multiphoton Excitation ◽  
Fluorescence Correlation ◽  
Large Stokes Shift

We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

scholarly journals Quantifying Diffusion in a Biofilm ofStreptococcus mutans

2010 ◽  
Vol 55 (3) ◽  
pp. 1075-1081 ◽  
Author(s):  
Zeshi Zhang ◽  
Elena Nadezhina ◽  
Kevin J. Wilkinson
Keyword(s):  
Ionic Strength ◽  
Fluorescent Probes ◽  
Diffusion Coefficients ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Self Diffusion ◽  
Probe Size ◽  
Order Of Magnitude ◽  
Biofilm Heterogeneity ◽  
Insight Into

ABSTRACTIn biofilms, diffusion may limit the chemical activity of nutrients, toxic compounds, and medicines. This study provides direct, noninvasive insight into the factors that will most effectively limit the transport of antibiotics and biocides in biofilms. Self-diffusion coefficients have been determined for a number of fluorescent probes in biofilms ofStreptococcus mutansusing fluorescence correlation spectroscopy. The effects of probe size and charge and the roles of biofilm pH, ionic strength, and heterogeneity were studied systematically. The relative diffusion coefficients (Din the biofilm divided by that in water) decreased with increasing probe size (3,000-molecular-weight [3K], 10K, 40K, 70K, and 2,000K dextrans). Studies using variably charged substrates (tetramethylrhodamine, Oregon Green, rhodamine B, and rhodamine 6G) showed that the self-diffusion coefficients decreased with an increasing negative charge of the fluorescent probes. No significant effect was observed for changes to the ionic strength (10−4to 10−1M) or pH (4 to 9) of the biofilm. Biofilm heterogeneity was responsible for variations of ca. one order of magnitude in the diffusion coefficients.

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