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10 ARTICLES PUBLISHED IN JoVE

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Biology

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography
Matthew C. Johnson *1, Frederik Rudolph *1,2, Tina M. Dreaden *3, Gengxiang Zhao *1, Bridgette A. Barry 3, Ingeborg Schmidt-Krey 1,3
1School of Biology, Georgia Institute of Technology, 2Department of Molecular Pharmacology, RWTH Aachen University, 3School of Chemistry and Biochemistry, Georgia Institute of Technology

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 – 90kDa.

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Immunology and Infection

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice
Anna Zychlinsky Scharff 1, Matthew L. Albert 1,2, Molly A. Ingersoll 1
1Unité d’Immunobiologie des Cellules Dendritiques, Department of Immunology, Institut Pasteur, INSERM U818, 2Genentech

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

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Immunology and Infection

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry
Ryan M. Baxter *1, Daniel S. Kong *1, Josselyn E. Garcia-Perez 1, William E. O'Gorman 2, Elena W.Y. Hsieh 1,3
1Department of Immunology and Microbiology, University of Colorado School of Medicine, 2OMNI Biomarkers, Development Sciences, Genentech, 3Department of Pediatrics, Division of Allergy and Immunology, University of Colorado School of Medicine

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

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Biochemistry

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions
Bushra Husain 1, Sairupa Paduchuri 1, Sree R. Ramani 2, Nadia Martinez-Martin 1
1Receptor Discovery group, Microchemistry, Proteomics and Lipidomics Department, Genentech, 2Portfolio Management and Operations, Genentech

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

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Biochemistry

gP2S, an Information Management System for CryoEM Experiments
Dorota Wypych 1, Daniel Kierecki 1, Filip M. Golebiowski 1, Alexis Rohou 2
1Roche Polska Sp. z o.o., 2Department of Structural Biology, Genentech

gP2S is a web application for the tracking of cryoEM experiments. Its main features are described, as are the steps required to install and configure the application. Once configured, the application allows one to accurately record metadata associated with negative stain and cryoEM experiments.

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Cancer Research

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues
Charles A. Havnar 1, Oliver Zill 2, Jeff Eastham 1, Jeffrey Hung 1, Manana Javey 3, Emmanuel Naouri 3, Jennifer Giltnane 1, Justin M. Balko 4, Andrew Wallace 2, Nicolas Lounsbury 2, Daniel Oreper 2, Sarajane Saturnio 1, G-Y Yang 5, Amy A. Lo 1
1Departments of Research Pathology, Genentech, 2Bioinformatics & Computational Biology, Genentech, 3Roche Sequencing Solutions, Hacienda Drive, 4Department of Medicine, Vanderbilt University Medical Center, Medical Center Drive, 5Department of Pathology, Northwestern University, Feinberg School of Medicine

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

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Cancer Research

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection
Veronica Ibarra-Lopez 1, Sangeeta Jayakar 1, Yeqing Angela Yang 2, Ciara Martin 3, Zora Modrusan 2, Sandra Rost 1
1Department of Pathology, Genentech, 2Department of Microchemistry, Proteomics, Lipidomics & Next Generation Sequencing (MPL-NGS), Genentech, 3NanoString Technologies

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 µm.

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Medicine

Synthetic Antigen Controls for Immunohistochemistry
Charles A. Havnar 1, Kathy J. Hötzel 1, Charles A. Jones 1, Carmina M. Espiritu 1, Linda K. Rangell 1, Franklin V. Peale 1
1Research Pathology, Genentech, Inc.

This work documents a simple method to create synthetic antigen controls for immunohistochemistry. The technique is adaptable to a variety of antigens in a wide range of concentrations. The samples provide a reference with which to assess intra- and inter-assay performance and reproducibility.

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Biochemistry

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples
Matthew C. Johnson *1,2, Arshay J. Grant *1, Ingeborg Schmidt-Krey 1,3
1School of Biological Sciences, Georgia Institute of Technology, 2Department of Structural Biology, Genentech, 3School of Chemistry and Biochemistry, Georgia Institute of Technology

The peel-blot technique is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

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Biology

Standardized Processing for Formalin-Fixed, Paraffin-Embedded Cell Pellet Immunohistochemistry Controls
Charles Havnar 1, Kathy Hotzel 1, Carmina Espiritu 1, Amy Lo 1, Joshua D. Webster 1
1Department of Pathology, Genentech

Presented here is a protocol for generating formalin-fixed, paraffin-embedded cell pellet controls for immunohistochemistry.

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