Molecular Biology

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    Protocols in Current Issue
    Isolation of Pure Mitochondria from Rat Kidneys and Western Blot of Mitochondrial Respiratory Chain Complexes
    [Abstract] Cardiac, neuronal and renal tubular epithelial cells are the most metabolically active cells in the body. Their fate depends largely on their mitochondria as the primary energy generating system which participates in the control of apoptosis, cell cycle and metabolism. Thus, mitochondrial dysfunction is a hallmark of many chronic diseases ...
    Identification of Heteroreceptors Complexes and Signal Transduction Events Using Bioluminescence Resonance Energy Transfer (BRET)
    Authors:  Irene Reyes-Resina, Jasmina Jiménez, Gemma Navarro and Rafael Franco, date: 10/05/2019, view: 161, Q&A: 0
    [Abstract] Detecting protein-protein interactions by co-immunoprecipitation provided a major advancement in the immunology research field. In the G-protein-coupled receptors (GPCRs) research field, colocalization and co-immunoprecipitation were used to detect interactions, but doubts arose due to specificity of the antibodies (monoclonal in the case of ...
    Analyzing the Functionality of Non-native Hsp70 Proteins in Saccharomyces cerevisiae
    Authors:  Laura E. Knighton, Lizbeth P. Saa, Adam M. Reitzel and Andrew W. Truman, date: 10/05/2019, view: 111, Q&A: 0
    [Abstract] Yeast are an ideal system to study Heat Shock Protein 70 (Hsp70) function in a cellular context. This protocol was generated to analyze the function of non-native Hsp70 proteins by expressing them as the sole cytosolic Hsp70 in yeast. As an initial step, Hsp70 variants (such as Ssa1 point mutants and non-yeast versions such as Nematostella ...
    A PhoA-STII Based Method for Efficient Extracellular Secretion and Purification of Fab from Escherichia coli
    Authors:  Ziyan Wang, Yang Gao, Manyu Luo, Cedric Cagliero, Hua Jiang, Yueqing Xie, Jianwei Zhu and Huili Lu, date: 09/20/2019, view: 205, Q&A: 0
    [Abstract] In comparison with full-length IgGs, antigen binding fragments (Fabs) are smaller in size and do not require the complexed post-translational modification. Therefore, Fab can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, the disulfide-bonds containing exogenous protein, including ...
    A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material
    [Abstract] We describe here a detailed, refined protocol for the generation of citrulline-specific monoclonal antibodies from single human B cells from rheumatoid arthritis (RA) patients. This protocol provides a detailed guide of the procedure starting from single B cells of your choice and followed by amplification of the variable region of immunoglobulin ...
    Three-dimensional Reconstruction and Quantification of Proteins and mRNAs at the Single-cell Level in Cultured Cells
    Authors:  Xueer Jiang, Ingrid Brust-Mascher and Li-En Jao, date: 08/20/2019, view: 653, Q&A: 0
    [Abstract] Gene expression is often regulated by the abundance, localization, and translation of mRNAs in both space and time. Being able to visualize mRNAs and protein products in single cells is critical to understand this regulatory process. The development of single-molecule RNA fluorescence in situ hybridization (smFISH) allows the detection of ...
    A Radioactive in vitro ERK3 Kinase Assay
    Authors:  Lobna Elkhadragy and Weiwen Long, date: 08/20/2019, view: 365, Q&A: 0
    [Abstract] Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that have an important role in signal transduction. Extracellular signal-regulated kinase 3 (ERK3), also known as MAPK6, is an atypical MAPK. Here, we describe in detail an in vitro assay for the kinase activity of ERK3 using myelin basic protein (MBP) or steroid ...
    In vitro RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay
    Authors:  G. Elif Karagöz, Jirka Peschek, Peter Walter and Diego Acosta-Alvear, date: 07/20/2019, view: 1111, Q&A: 0
    [Abstract] The kinase/RNase IRE1 is a key effector of the cellular response to endoplasmic reticulum stress. The RNase activity of IRE1 can be measured in cells or in the test tube. Here we describe a protocol for the in vitro cleavage and analysis of RNA substrates of IRE1. The method consists of the in vitro transcription, purification ...
    Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro
    [Abstract] The stability of the HIV-1 capsid and the spatiotemporal control of its disassembly, a process called uncoating, need to be finely tuned for infection to proceed. Biochemical methods for measuring capsid lattice disassembly in bulk are unable to resolve intermediates in the uncoating reaction. We have developed a single-particle fluorescence ...
    In vitro and in vivo Assessment of Protein Acetylation Status in Mycobacteria
    Authors:  Krishna K. Singh, Devendra P. Singh, Rambir Singh and Deepak K. Saini, date: 07/05/2019, view: 647, Q&A: 0
    [Abstract] Protein acetylation is one of the standard post-translational modifications found in proteins across all organisms, along with phosphorylation which regulates diverse cellular processes. Acetylation of proteins can be enzymatically catalyzed through acetyltransferases, acetyl CoA synthetases or non-enzymatically through acyl carrier metabolic ...



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