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Wesleyan University

8 ARTICLES PUBLISHED IN JoVE

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JoVE Journal

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
F. Noah Biro 1, Jie Zhai 1, Christopher W. Doucette 1, Manju M. Hingorani 1
1Molecular Biology and Biochemistry Department, Wesleyan University

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.

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Developmental Biology

Live-imaging of the Drosophila Pupal Eye
Mark B. Hellerman 1, Richard H. Choe 1, Ruth I. Johnson 1
1Biology Department, Wesleyan University

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

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Education

Methods for Measuring the Orientation and Rotation Rate of 3D-printed Particles in Turbulence
Brendan C. Cole 1, Guy G. Marcus 1, Shima Parsa 1, Stefan Kramel 1, Rui Ni 1, Greg A. Voth 1
1Department of Physics, Wesleyan University

We use 3D printing to fabricate anisotropic particles in the shapes of jacks, crosses, tetrads, and triads, whose alignments and rotations in turbulent fluid flow can be measured from multiple simultaneous video images.

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Biochemistry

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition
Stacy N. Uchendu 1, Angelika Rafalowski 1, Erin F. Cohn 1, Luke W. Davoren 1, Erika A. Taylor 1
1Department of Chemistry, Wesleyan University

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

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Developmental Biology

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation
Miles W. DeAngelis 1, Ruth I. Johnson 1
1Department of Biology, Wesleyan University

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction. 

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Biochemistry

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
Candice M. Etson 1,2, Petar Todorov 1, Nooshin Shatery Nejad 2, Nirmala Shrestha 2, David R. Walt 1,3
1Department of Chemistry, Tufts University, 2Department of Physics, Wesleyan University, 3Wyss Institute at Harvard University

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

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Biochemistry

Förster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features
Jack Northrop 1, Donald B. Oliver 1,2, Ishita Mukerji 1,2
1Molecular Biology and Biochemistry Department, Wesleyan University, 2Molecular Biophysics Program, Wesleyan University

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

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Reconstructing Terrestrial Paleoclimate and Paleoecology with Fossil Leaves Using Digital Leaf Physiognomy and Leaf Mass Per Area

Reconstructing Terrestrial Paleoclimate and Paleoecology with Fossil Leaves Using Digital Leaf Physiognomy and Leaf Mass Per Area
Alexander J. Lowe 1, Andrew G. Flynn 2, Matthew J. Butrim 3, Aly Baumgartner 4, Dana L. Royer 5, Daniel J. Peppe 6
1Department of Biology, University of Washington, 2Department of Paleobiology, National Museum of Natural History, Smithsonian Institution, 3Department of Geology and Geophysics, Program in Ecology, University of Wyoming, 4Department of Ecology and Evolutionary Biology, University of Michigan Herbarium, 5Department of Earth and Environmental Sciences, Wesleyan University, 6Department of Geosciences, Baylor University

The protocol presented shows digital measurement and analysis of continuous leaf physiognomic traits on fossil leaves to reconstruct paleoclimate and paleoecology using the digital leaf physiognomy and leaf mass per area reconstruction methods.

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