Protocols in Current Issue
    Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography
    [Abstract] We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR ...
    Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry
    Authors:  Manish Bhuwan, Madhuri Suragani, Nasreen Z. Ehtesham and Seyed E. Hasnain, date: 01/20/2017, view: 8089, Q&A: 0
    [Abstract] The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway ...
    Substituted Cysteine Accessibility Method for Topology and Activity Studies of Membrane Enzymes Forming Thioester Acyl Intermediates in Bacteria
    Authors:  Sébastien Gélis-Jeanvoine and Nienke Buddelmeijer, date: 11/05/2015, view: 6433, Q&A: 0
    [Abstract] The topology of membrane proteins and enzymes can be determined using various methods including reporter protein fusions and accessibility of cysteine residues to alkylating agents. Here we describe a variation of the substituted cysteine accessibility method to determine membrane topology and activity of enzymes containing an active site ...
    Semi-denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE)
    Authors:  Laura Molina-García and Fátima Gasset-Rosa, date: 11/20/2014, view: 15054, Q&A: 2
    [Abstract] Pathological proteins in neurodegenerative diseases suffer a conformational change to a misfolded amyloid state. Such pathological event leads to the aggregation of these proteins that indefinitely propagates as an altered form of itself, and harbor prion-like properties (Wickner, 1994; Prusiner, 2012). In addition to diseases, prions can also ...
    Zonal Sedimentation Analysis on Sucrose Gradients
    Author:  Javier G. Magadán, date: 04/20/2014, view: 7195, Q&A: 0
    [Abstract] Zonal sedimentation analysis on sucrose gradients allows estimation of the molecular size of an individual protein or a protein complex by centrifugation at a constant speed under nondenaturing conditions. This method is particularly suitable for globular proteins like the influenza A virus (IAV) protein hemagglutinin (HA). Here, I describe step ...

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