Protocols in Current Issue
    Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay
    Authors:  Ana Lechuga, Mónica Berjón-Otero, Margarita Salas and Modesto Redrejo-Rodríguez, date: 01/05/2018, view: 6963, Q&A: 0
    [Abstract] This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules ...
    Chase Assay of Protein Stability in Haloferax volcanii
    Authors:  Xian Fu and Julie A. Maupin-Furlow, date: 03/20/2017, view: 5253, Q&A: 0
    [Abstract] Highly regulated and targeted protein degradation plays a fundamental role in almost all cellular processes. Determination of the protein half-life by the chase assay serves as a powerful and popular strategy to compare the protein stability and study proteolysis pathways in cells. Here, we describe a chase assay in Haloferax volcanii, a ...
    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: 5712, 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 ...
    Preparation of Parasite Protein Extracts and Western Blot Analysis
    Authors:  Arlett Heiber and Tobias Spielmann, date: 06/05/2014, view: 20892, Q&A: 0
    [Abstract] In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, different methods are available ...
    Western Blotting for Staphylococcus aureus AgrA
    Authors:  Chikara Kaito and Kazuhisa Sekimizu, date: 03/20/2014, view: 10712, Q&A: 0
    [Abstract] Staphylococcus aureus has a quorum sensing system to regulate the expression of various virulence factors, which is exerted by the agr locus that encodes agrBDCA and a regulatory RNA called RNAIII. AgrB, AgrD, and AgrC proteins are involved in producing and recognizing extracellular quorum sensing molecules and transduce ...
    Co-immunoprecipitation of Flag-TLR3 or Myc-MSR1 with HCV RNA
    Authors:  Daisuke Yamane, Hiromichi Dansako and Stanley M. Lemon, date: 03/05/2014, view: 7992, Q&A: 0
    [Abstract] Co-immunoprecipitation assay of TLR3-Flag or Myc-MSR1 with HCV RNA is used to identify direct interaction of viral RNA with host proteins that recognize viral RNA to initiate interferon (IFN) signaling, a crucial antiviral response of the host cells. Both Toll-like receptor 3 (TLR3) and class-A scavenger receptor type 1 (MSR1) proteins recognize ...
    Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress
    Authors:  Fiona Hackett, Christine R Collins, Malcolm Strath and Michael J Blackman, date: 03/05/2014, view: 8784, Q&A: 0
    [Abstract] Upon rupture of Plasmodium falciparum (P. falciparum) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the merozoite surface. ...
    Shigella IpaD and IpaB Surface Localizations
    Authors:  Lionel Schiavolin, Alaeddine Meghraoui and Abdelmounnaim Allaoui, date: 11/20/2013, view: 8773, Q&A: 1
    [Abstract] Shigella uses a type III secretion system to invade host cell and to cause disease. Secretion control and insertion of a translocation pore into cell membrane are critical steps for pathogenesis and are tightly linked to the formation of the needle tip complex formed by the IpaB and IpaD proteins (Veenendaal et al., 2007). ...
    Immunoblot Analysis of Histone H4 Acetylation and Histone H2A Phosphorylation in Candida albicans
    Authors:  Michael Tscherner and Karl Kuchler, date: 10/20/2013, view: 7813, Q&A: 0
    [Abstract] Posttranslational modifications of histones are required for different processes including transcription, replication and DNA damage repair. This protocol describes the preparation of a whole-cell extracts for the fungal pathogen Candida albicans. Furthermore, the extract is used to detect lysine acetylation of histone H4 as well as ...
    Virus Overlay Assay (Far-Western blotting)
    Authors:  Kun-Tong Jia, Chang-Jun Guo, Xiao-Bo Yang and Jian-Guo He, date: 10/05/2013, view: 12877, Q&A: 0
    [Abstract] Virus overlay assay is a method to detect protein-protein interaction in vitro. We performed the virus overlay assay to identify the receptor proteins interacting with the infectious spleen and kidney necrosis virus (ISKNV).

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