Accedi

ShanghaiTech University

10 ARTICLES PUBLISHED IN JoVE

Immunology and Infection

Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries

Shane Miersch^1,2, Zhijian Li^1,2, Rachel Hanna^1,2, Megan E. McLaughlin^1,2, Michael Hornsby^1,3, Tet Matsuguchi^1,3, Marcin Paduch^1,4, Annika Sääf^1,4, Jim Wells^1,3, Shohei Koide^1,4, Anthony Kossiakoff^1,4, Sachdev S. Sidhu^1,2

¹The Recombinant Antibody Network, ²The Banting and Best Department of Medical Research, University of Toronto, ³Antibiome Center, University of California, San Francisco at Mission Bay, ⁴Department of Biochemistry and Molecular Biology, The University of Chicago

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys₂-His₂ Zinc-finger Domains

Thomas Gaj^*1, Jia Liu^*1,2

¹Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute, ²Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Neuroscience

Optogenetic Functional MRI

Peter Lin¹, Zhongnan Fang², Jia Liu¹, Jin Hyung Lee^1,2

¹Neurology and Neurological Sciences, Stanford University, ²Electrical Engineering, Neurology and Neurological Sciences, Stanford University

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

Developmental Biology

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

Bao Li Zhang^1,2,3, Da Yuan Gao^1,2,3, Xiao Xu Zhang^1,3, Shuo Shi⁴, Winnie Shum¹

¹School of Life Science and Technology, ShanghaiTech University, ²Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, ³University of Chinese Academy of Sciences, ⁴Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

Bioengineering

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Dong Wei^1,2, Jinjie Gu¹, Zhongxi Zhang¹, Chenhong Wang¹, Dexin Wang¹, Chul Ho Kim³, Biao Jiang¹, Jiping Shi^1,4, Jian Hao¹

¹Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, ²University of Chinese Academy of Sciences, ³Biorefinery Research Center, Jeonbuk Branch Institute, Korea Research Institute of Bioscience & Biotechnology (KRIBB), ⁴School of Life Science and Technology, ShanghaiTech University

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

Immunology and Infection

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Ganggang Huang^*1, Zhenwei Zhong^*1, Shane Miersch^2,3, Sachdev S. Sidhu^1,2,3, Shin-chen Hou¹, Donghui Wu¹

¹Laboratory of Antibody Engineering, Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, ²Banting and Best Department of Medical Research, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto, ³Department of Molecular Genetics, Terrence Donnelly Center for Cellular and Biomolecular Research, University of Toronto

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

Genetics

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Lina Yu^*1, Wenxiu He^*1, Jie Xie^*1, Rui Guo^*1, Juan Ni², Xia Zhang³, Quishi Xu¹, Caifeng Wang¹, Qiuling Yue¹, Fangfang Li¹, Mengcheng Luo⁴, Bo Sun³, Lan Ye¹, Ke Zheng¹

¹State Key Laboratory of Reproductive Medicine, Nanjing Medical University, ²The Affiliated Hospital of Hangzhou Normal University, ³School of Life Science and Technology, ShanghaiTech University, ⁴Department of Tissue and Embryology, School of Basic Medical Sciences, Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely applicable for studying protein-nucleic acid interactions.

Chemistry

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

Weng-I Katherine Chio¹, Gemma Davison¹, Tabitha Jones¹, Jia Liu¹, Ivan P. Parkin¹, Tung-Chun Lee^1,2

¹Department of Chemistry, University College London (UCL), ²Institute for Materials Discovery, University College London (UCL)

A host-guest complex of cucurbit[7]uril and uric acid was formed in an aqueous solution before adding a small amount into Au NP solution for quantitative surface-enhanced Raman spectroscopy (SERS) sensing using a modular spectrometer.

Biochemistry

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Jia Liu^*1, Yuyang Liu^*1, Weidi Chen¹, Yuzhen Ding¹, Xiaoru Lan¹, Wei Liu¹

¹Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

Cancer Research

An R-Based Landscape Validation of a Competing Risk Model

Haiping Lin^*1,3, Hongjuan Zheng^*2, Chenyang Ge³, Lixian Ling², Ruihua Yin², Qinghua Wang², Xia Zhang², Shishi Zhou², Xiayun Jin², Xifeng Xu², Jianfei Fu²

¹Department of Hepatobiliary Surgery, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, ²Department of Medical Oncology, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, ³Department of Colorectal Surgery, Affiliated Jinhua Hospital, Zhejiang University School of Medicine

The present protocol describes codes in R for evaluating the discrimination and calibration abilities of a competing risk model, as well as codes for the internal and external validation of it.