Protocols in Current Issue
    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: 486, 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: 351, 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: 1157, 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: 1736, 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: 1976, 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 ...
    Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
    Authors:  Jennifer Hirst, Robert R Kay and David Traynor, date: 06/05/2015, view: 4109, Q&A: 0
    [Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells ...
    Cyst Detection in Toxoplasma gondii Infected Mice and Rats Brain
    [Abstract] Toxoplasmosis caused by the intracellular parasite Toxoplasma gondii, is characterized by a life-long chronic infection. The parasite is an efficient neurotropic infectious agent that establishes its “safe” life by forming intracellular cysts in chronically infected animals and humans. This protocol describes the specific recipes and ...
    A Gas Chromatography-Mass Spectrometry-Based Two Stage Assay for Measurement of in vitro myo-Inositol 3-phosphate Synthase (INO1) Activity
    Authors:  James I. MacRae and Malcolm J. McConville, date: 03/05/2015, view: 3195, Q&A: 0
    [Abstract] This method describes an in vitro assay for measuring INO1 enzyme activity (the conversion of glucose 6-phosphate to myo-inositol 3-phosphate) in cell-free extracts. The method was first described for Plasmodium falciparum cells in MacRae et al. (2014) and consists of two parts: Part 1 describes the assay itself ...
    Preparation of Parasite Protein Extracts and Western Blot Analysis
    Authors:  Arlett Heiber and Tobias Spielmann, date: 06/05/2014, view: 9018, 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 ...
    Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites
    Authors:  Arlett Heiber, Silke Retzlaff and Tobias Spielmann, date: 06/05/2014, view: 3378, Q&A: 0
    [Abstract] This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission ...