Protocols in Current Issue
    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 ...
    Coupling of HIV-1 gp120-derived Core Protein to Paramagnetic Beads and Adsorption Assays
    Authors:  Jidnyasa Ingale and Richard T Wyatt, date: 10/05/2015, view: 6270, Q&A: 0
    [Abstract] Analysis of the functional activity in polyclonal serum following immunization of a complex protein or glycoprotein immunogen is a very important but tedious process. Fine mapping of epitope-specific antibodies is difficult when they are elicited at relatively low levels. In our recent study focused on developing an HIV-1 vaccine, we immunized ...
    Kinetic Analysis of Monoclonal Antibody Binding to HIV-1 gp120-derived Hyperglycosylated Cores
    Authors:  Jidnyasa Ingale and Richard T Wyatt, date: 10/05/2015, view: 7340, Q&A: 0
    [Abstract] Kinetic analysis of antibodies is one of the important studies for characterization of antibodies and screening of ligands. In our recent study (Ingale et al., 2014), we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer ...
    RNase H Polymerase-independent Cleavage Assay for Evaluation of RNase H Activity of Reverse Transcriptase Enzymes
    Authors:  Angela Corona and Enzo Tramontano, date: 08/20/2015, view: 11695, Q&A: 0
    [Abstract] The ribonuclease H (RNase H) polymerase-independent cleavage assay allows detection and quantification of RNase H activity of reverse transcriptase (RT) enzymes with a hybrid substrate formed by a fluorescein labeled RNA annealed with Dabcyl DNA (Figure 1). Here we describe a protocol that we have adapted for HIV-1 RT expressed from a ...
    Mismatched Primer Extension Assays
    Authors:  Vasudevan Achuthan and Jeffrey J. DeStefano, date: 06/20/2015, view: 6700, Q&A: 0
    [Abstract] Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3ʹ end is a major strength of the mismatched primer extension assays. Recently, we used ...
    Primer Extension Reactions for the PCR- based α- complementation Assay
    Authors:  Vasudevan Achuthan and Jeffrey J. DeStefano, date: 06/20/2015, view: 9623, Q&A: 0
    [Abstract] The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus ...
    HIV-1 Fusion Assay
    Authors:  Marielle Cavrois, Jason Neidleman and Warner C. Greene, date: 08/20/2014, view: 14367, Q&A: 0
    [Abstract] The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase–Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a ...
    HIV-1 Virus-like Particle Budding Assay
    Authors:  Nathan H Vande Burgt, Luis J Cocka and Paul Bates, date: 07/20/2013, view: 9752, Q&A: 0
    [Abstract] Viral replication culminates with the egress of the mature virion from the host cell. This step of the viral life cycle has recently garnered increased attention with the discovery of the cellular restriction factor, Tetherin, which tethers budded virions to the surface of infected cells and inhibits viral spread. The importance of this block in ...

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