分子生物学

分类

    现刊
    Measurement of LRRK2 Kinase Activity by Proximity Ligation Assay
    通过邻近连接测定法测量 LRRK2 激酶活性
    作者:Matthew T. Keeney, Eric K. Hoffman, J. Timothy Greenamyre and Roberto Di Maio日期:09/05/2021,浏览量:438,Q&A: 0
    [Abstract]

    Missense mutations in leucine rich-repeat kinase 2 (LRRK2) cause forms of familial Parkinson’s disease and have been linked to ‘idiopathic’ Parkinson’s disease. Assessment of LRRK2 kinase activity has been very challenging due to its size, complex structure, and relatively low abundance. A standard in the field to assess

    ...
    Kinetic Analysis of a Protein-protein Complex to Determine its Dissociation Constant (KD) and the Effective Concentration (EC50) of an Interplaying Effector Molecule Using Bio-layer Interferometry
    蛋白质-蛋白质复合物的动力学分析,以确定其电离常数 (KD) 和使用生物层干涉法相互作用的效应分子的有效浓度 (EC50)
    作者:Tim Orthwein, Luciano F. Huergo, Karl Forchhammer and Khaled A. Selim日期:09/05/2021,浏览量:954,Q&A: 0
    [Abstract]

    Biolayer interferometry (BLI) is an emerging analytical tool that allows the study of protein complexes in real time to determine protein complex kinetic parameters. This article describes a protocol to determine the KD of a protein complex using a 6×His tagged fusion protein as bait immobilized on the NTA sensor chip of the

    ...
    Optimised Method for the Production and Titration of Lentiviral Vectors Pseudotyped with the SARS-CoV-2 Spike
    SARS-CoV-2刺突慢病毒伪型载体制备和滴定的优化方法
    [Abstract]

    The use of recombinant lentivirus pseudotyped with the coronavirus Spike protein of SARS-CoV-2 would circumvent the requirement of biosafety-level 3 (BSL-3) containment facilities for the handling of SARS-CoV-2 viruses. Herein, we describe a fast and reliable protocol for the transient production of lentiviruses pseudotyped with SARS-CoV-2 Spike

    ...
    Monitoring Protein Splicing Using In-gel Fluorescence Immediately Following SDS-PAGE
    SDS-PAGE后立即使用凝胶内荧光监测蛋白质剪接
    作者:Joel Weinberger II and Christopher W. Lennon日期:08/20/2021,浏览量:1139,Q&A: 0
    [Abstract]

    Inteins garner significant interest from both basic and applied researchers due to their unique catalytic abilities. Herein, we describe a protocol for accurately monitoring protein splicing without purification using in-gel fluorescence immediately following Tris-Glycine SDS-PAGE. Following expression in Escherichia coli, cells are lysed by

    ...
    Unraveling the Physicochemical Determinants of Protein Liquid-liquid Phase Separation by Nanoscale Infrared Vibrational Spectroscopy
    利用纳米尺度红外振动光谱揭示蛋白质液-液相分离的理化决定因素
    作者:Francesco S. Ruggeri, Alyssa M. Miller, Michele Vendruscolo and Tuomas P. J. Knowles日期:08/20/2021,浏览量:876,Q&A: 0
    [Abstract]

    The phenomenon of reversible liquid-liquid phase separation of proteins underlies the formation of membraneless organelles, which are crucial for cellular processes such as signalling and transport. In addition, it is also of great interest to uncover the mechanisms of further irreversible maturation of the functional dense liquid phase into

    ...
    Synchronized Real-time Measurement of Sec-mediated Protein Translocation
    Sec介导的蛋白质易位的同步实时测量
    作者:Riti Gupta, Dmitri Toptygin and Christian M. Kaiser日期:08/20/2021,浏览量:647,Q&A: 0
    [Abstract]

    The Sec translocon, consisting of a heterotrimeric transmembrane channel (SecYEG) and an associated ATPase (SecA), catalyzes the export of unfolded proteins from the cytosol in bacteria. Kinetically resolving protein translocation at high resolution yields mechanistic insight into the process. Translocation is typically followed by measuring the

    ...
    Fe-NTA Microcolumn Purification of Phosphopeptides from Immunoprecipitation (IP) Eluates for Mass Spectrometry Analysis
    Fe-NTA微柱纯化免疫沉淀(IP)洗脱物中磷酸肽的质谱分析
    作者:Ethan J. Sanford and Marcus B. Smolka日期:08/05/2021,浏览量:503,Q&A: 0
    [Abstract]

    Protein phosphorylation is a nearly universal signaling mechanism. To date, a number of proteomics tools have been developed to analyze phosphorylation. Phosphoproteome-wide analyses using whole cell extracts suffer from incomplete coverage, often missing phosphorylation events from low-abundance proteins. In order to increase coverage of

    ...
    A Simple Method to Generate Super-sensitive AID (ssAID)-based Conditional Knockouts using CRISPR-based Gene Knockout in Various Vertebrate Cell Lines
    小胶质细胞的分离及蛋白表达的流式细胞仪分析:避免小胶质细胞背景荧光的陷阱
    作者:Kohei Nishimura and Tatsuo Fukagawa日期:07/20/2021,浏览量:705,Q&A: 0
    [Abstract]

    Inducing loss of function of a target protein using methods such as gene knockout is a powerful and useful strategy for analyzing protein function in cells. In recent years, the CRISPR/Cas-9-based gene knockout technology has been widely used across a variety of eukaryotes; however, this type of simple gene knockout strategy is not applicable to

    ...
    Differential Analysis of N-glycopeptide Abundance and N-glycosylation Site Occupancy for Studying Protein N-glycosylation Dysregulation in Human Disease
    用于研究人类疾病中蛋白质N-糖基化失调的N-糖肽丰度和N-糖基化位点的差异分析
    作者:Qi Zhang, Cheng Ma, Lian Li and Lih-Shen Chin日期:06/20/2021,浏览量:1069,Q&A: 0
    [Abstract]

    Protein N-glycosylation plays a vital role in diverse cellular processes, and dysregulated N-glycosylation is implicated in a variety of human diseases including neurodegenerative disorders and cancer. With recent advances in high-resolution mass spectrometry-based glycoproteomics technologies enabling large-scale N-glycoproteome profiling of

    ...
    Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension
    利用悬浮培养的人胚胎肾(HEK293)细胞优化分泌蛋白的重组生产
    [Abstract]

    Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In

    ...