Fluorescence Correlation and Cross-Correlation Spectroscopy Using Fluorescent Proteins for Measurements of Biomolecular Processes in Living Organisms

2011 ◽  
pp. 213-248 ◽  
Author(s):  
Yong Hwee Foo ◽  
Vladimir Korzh ◽  
Thorsten Wohland
Keyword(s):  
Cross Correlation ◽  
Fluorescent Proteins ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Living Organisms

scholarly journals Fluorescence Correlation and Cross-Correlation Spectroscopy Unveil Cytoplasmic mRNP Composition and Dynamics

2020 ◽  
Author(s):  
Àngels Mateu-Regué ◽  
Jan Christiansen ◽  
Christian Hellriegel ◽  
Finn Cilius Nielsen
Keyword(s):  
Life Cycle ◽  
Dynamic Analysis ◽  
Cross Correlation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Macromolecular Complexes ◽  
Fluorescence Correlation ◽  
Macromolecular Composition

ABSTRACTUnderstanding the mRNA life cycle requires analysis of the dynamic macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study mRNP complexes because they readily provide information about the molecular composition, stoichiometry, heterogeneity and dynamics of the particles. We developed FCS protocols for analysis of live cells and cellular lysates, and demonstrate the feasibility of analysing common cytoplasmic mRNPs composed of core factor YBX1, IMPs (or IGF2BPs) and their interactions with other RNA binding proteins such as PABPC1, ELAVL2 (HuB), STAU1 and FMRP. FCCS corroborated previously reported RNA dependent interactions between the factors and provided an estimate of the relative overlap between the factors in the mRNPs. In this way FCS and FCCS provide a new and useful approach for the quantitative and dynamic analysis of mRNP macromolecular complexes that may complement current biochemical approaches.

scholarly journals Photodynamics of Red Fluorescent Proteins Studied by Fluorescence Correlation Spectroscopy

2004 ◽  
Vol 86 (1) ◽  
pp. 384-394 ◽  
Author(s):  
Andreas Schenk ◽  
Sergey Ivanchenko ◽  
Carlheinz Röcker ◽  
Jörg Wiedenmann ◽  
G. Ulrich Nienhaus
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Fluorescent Proteins ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Red Fluorescent Proteins

Properties of Baculovirus Particles Displaying GFP Analyzed by Fluorescence Correlation Spectroscopy

2002 ◽  
Vol 383 (12) ◽  
pp. 1941-1946 ◽  
Author(s):  
J. Toivola ◽  
K. Ojala ◽  
P.O. Michel ◽  
M. Vuento ◽  
C. Oker-Blom
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Sodium Dodecyl ◽  
Recombinant Baculovirus ◽  
Mean Value ◽  
Correlation Spectroscopy ◽  
Viral Envelope ◽  
Fluorescence Correlation

Abstract Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89±0.74)10 8 cm2s 1 and an apparent hydrodynamic radius of 83.35±21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.5710 7 cm2s 1), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.

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