Protocols in Current Issue
    CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii
    Authors:  Shaojun Long, Kevin M. Brown and L. David Sibley, date: 03/20/2018, view: 453, Q&A: 0
    [Abstract] Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii. When combined with CRISPR/Cas9 based tagging, this ...
    Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii
    Authors:  Kevin M. Brown, Shaojun Long and L. David Sibley, date: 02/20/2018, view: 751, Q&A: 0
    [Abstract] Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii’s 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to ...
    MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes
    Authors:  Maximilian G. Plach, Klaus Grasser and Thomas Schubert, date: 12/05/2017, view: 1460, Q&A: 0
    [Abstract] Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides ...
    Drosophila Model of Leishmania amazonensis Infection
    Authors:  Kendi Okuda and Neal Silverman, date: 12/05/2017, view: 788, Q&A: 0
    [Abstract] This protocol describes how to generate and harvest antibody-free L. amazonensis amastigotes, and how to infect adult Drosophila melanogaster with these parasites. This model recapitulates key aspects of the interactions between Leishmania amastigotes and animal phagocytes.
    Quantification of Trypanosoma cruzi in Tissue and Trypanosoma cruzi Killing Assay
    Authors:  Hisako Kayama, Shoko Kitada and Kiyoshi Takeda, date: 11/20/2017, view: 911, Q&A: 0
    [Abstract] Infection with Trypanosoma cruzi causes Chagas disease. The methods provided here allow for the quantification of T. cruzi in the liver, heart, and blood of intraperitoneally-infected mice and analysis of the killing activity of the cells infected with T. cruzi in vitro.
    Liposome Disruption Assay to Examine Lytic Properties of Biomolecules
    Authors:  John R. Jimah, Paul H. Schlesinger and Niraj H. Tolia, date: 08/05/2017, view: 2116, Q&A: 0
    [Abstract] Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we ...
    Plasmodium Sporozoite Motility on Flat Substrates
    Authors:  Henriette L Prinz, Julia M Sattler and Friedrich Frischknecht, date: 07/20/2017, view: 1426, Q&A: 0
    [Abstract] Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro.
    Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
    Authors:  Noelia Lander, Miguel A. Chiurillo, Aníbal E. Vercesi and Roberto Docampo, date: 05/20/2017, view: 3354, Q&A: 0
    [Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then ...
    Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays
    Authors:  Ashwin D’Souza, Paulomi Sanghavi, Ashim Rai, Divya Pathak and Roop Mallik, date: 12/05/2016, view: 2926, Q&A: 0
    [Abstract] We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined ...
    In vitro Fluorescent Matrix Degradation Assay for Entamoeba histolytica
    Authors:  Merlyn Emmanuel and Sunando Datta, date: 03/20/2016, view: 2894, Q&A: 0
    [Abstract] Fluorescent matrix degradation assay is a popular and widely used assay in the field of invadopodium biology (Artym et al., 2009). Matrix remodeling and degradation can be observed under both physiological and pathological conditions. Cancer cells extensively remodel and degrade the underlying matrix by employing actin-rich protrusive ...