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State University of New York

9 ARTICLES PUBLISHED IN JoVE

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Biology

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment
Shoko Ueki 1, Benjamin L. Meyers 1, Farzana Yasmin 2, Vitaly Citovsky 2
1Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, 2Bio-Medical Engineering Department, NED University of Engineering and Technology

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

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Biology

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster
Aaron T. Haselton 1, Yih-Woei C. Fridell 2
1Department of Biology, State University of New York, 2Allied Health Sciences, University of Connecticut

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

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Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
Shoko Ueki 1, Benoît Lacroix 1, Vitaly Citovsky 1
1Department of Biochemistry and Cell Biology, State University of New York

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

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Biology

Assaying Proteasomal Degradation in a Cell-free System in Plants
Elena García-Cano 1, Adi Zaltsman 1, Vitaly Citovsky 1
1Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular “tags” for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

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Immunology and Infection

Identification of Plasmodesmal Localization Sequences in Proteins In Planta
Cheng Yuan 1, Sondra G. Lazarowitz 2, Vitaly Citovsky 1
1Department of Biochemistry and Cell Biology, State University of New York, 2Department of Plant Pathology and Plant-Microbe Biology, Cornell University

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

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Bioengineering

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
Minakshi Mukherjee *1, Emily Caroll *1, Zhen Q. Wang 1
1Department of Biological Sciences, University at Buffalo, State University of New York

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

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Bioengineering

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy
Yuhao Yuan 1, Fake Lu 1
1Department of Biomedical Engineering, Binghamton University, State University of New York

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

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Biology

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer
Mi Sa Vo Phan 1, Phu Tri Tran 1, Vitaly Citovsky 1
1Department of Biochemistry and Cell Biology, State University of New York

Fluorescence resonance energy transfer (FRET) is an imaging technique for detecting protein interactions in living cells. Here, a FRET protocol is presented to study the association of histone-modifying enzymes with transcription factors that recruit them to the target promoters for epigenetic regulation of gene expression in plant tissues.

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Environment

Measuring Dissolved Methane in Aquatic Ecosystems Using An Optical Spectroscopy Gas Analyzer
Jorge A. Villa 1, Robert Bordelon 1, Yang Ju 2, Madeline Moore 1, Timothy Morin 3, Gil Bohrer 2
1School of Geosciences, University of Louisiana at Lafayette, 2Department of Civil, Environmental and Geodetic Engineering, The Ohio State University, 3Department of Environmental Resources Engineering, College of Environmental Sciences and Forestry, State University of New York

This study demonstrates an approach to measure methane gas concentrations in aqueous samples using portable optical analyzers coupled to an injection chamber in a closed loop. The results are similar to conventional gas chromatography, presenting a practical and low-cost alternative particularly suitable for remote field studies.

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