State University of New York View Institution's Website 14 articles published in JoVE Biochemistry High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography Gabrielle R. Budziszewski1, M. Elizabeth Snell1, Tiffany R. Wright1, Miranda L. Lynch1, Sarah E. J. Bowman1,2 1National High-Throughput Crystallization Center, Hauptman-Woodward Medical Research Institute, 2Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, The State University of New York This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions. Biology Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer Mi Sa Vo Phan1, Phu Tri Tran1, Vitaly Citovsky1 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. Bioengineering A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy Yuhao Yuan1, Fake Lu1 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. Developmental Biology Primary Cell Cultures to Study the Regeneration Potential of Murine Müller Glia after MicroRNA Treatment Seoyoung Kang1, Stefanie G. Wohl1 1Department of Biological and Vision Sciences, College of Optometry, The State University of New York Müller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches. Chemistry ARL Spectral Fitting as an Application to Augment Spectral Data via Franck-Condon Lineshape Analysis and Color Analysis William R. Roberts1,2, Thomas N. Rohrabaugh1, Ryan M. O'Donnell1 1DEVCOM Army Research Laboratory (ARL), 2Department of Chemistry, State University of New York at Buffalo This protocol introduces Franck-Condon Lineshape Analyses (FCLSA) of emission spectra and serves as a tutorial for the use of ARL Spectral Fitting software. The open-source software provides an easy and intuitive way to perform advanced analysis of emission spectra including excited state energy calculations, CIE color coordinate determination, and FCLSA. Bioengineering Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning Minakshi Mukherjee*1, Emily Caroll*1, Zhen Q. Wang1 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. Immunology and Infection Identification of Plasmodesmal Localization Sequences in Proteins In Planta Cheng Yuan1, Sondra G. Lazarowitz2, Vitaly Citovsky1 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. Engineering Fabrication of Uniform Nanoscale Cavities via Silicon Direct Wafer Bonding Stephen R. D. Thomson1, Justin K. Perron2,3, Mark O. Kimball4, Sarabjit Mehta5, Francis M. Gasparini1 1Department of Physics, The State University of New York at Buffalo, 2Joint Quantum Institute, University of Maryland, 3The National Institute of Standards and Technology, 4Cryogenics and Fluids Branch, NASA Goddard Space Flight Center, 5HRL Laboratories A method for permanently bonding two silicon wafers so as to realize a uniform enclosure is described. This includes wafer preparation, cleaning, RT bonding, and annealing processes. The resulting bonded wafers (cells) have uniformity of enclosure ~1%1,2. The resulting geometry allows for measurements of confined liquids and gasses. Biology Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy Xiaowen Wang1, MacMillan Mbantenkhu1, Sara Wierzbicki1, Xin Jie Chen1 1Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography. Biology Genetic Modification and Recombination of Salivary Gland Organ Cultures Sharon J. Sequeira*1, Elise M. Gervais*1, Shayoni Ray1, Melinda Larsen1 1Department of Biological Sciences, University at Albany, SUNY A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors. Neuroscience Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing Ewan C. McNay1, Leslie A. Sandusky1, Jiah Pearson-Leary1 1Behavioral Neuroscience, University at Albany Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals. Neuroscience Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping N. Jeremy Hill1, Disha Gupta1,2, Peter Brunner1,2, Aysegul Gunduz1,2, Matthew A. Adamo3, Anthony Ritaccio2, Gerwin Schalk1,2,4,5,6,7 1Wadsworth Center, New York State Department of Health, 2Department of Neurology, Albany Medical College, 3Department of Neurosurgery, Albany Medical College, 4Department of Neurosurgery, Washington University, 5Department of Biomed. Eng., Rensselaer Polytechnic Institute, 6Department of Biomed. Sci., State University of New York at Albany, 7Department of Elec. and Comp. Eng., University of Texas at El Paso We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping. Biology Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro Shoko Ueki1, Benoît Lacroix1, Vitaly Citovsky1 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. Biology Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster Aaron T. Haselton1, Yih-Woei C. Fridell2 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.