Subcellular Fractionation of HA-GiCHMP7 Trophozoites

Crude subcellular fractionation experiment was performed on HA-GiCHMP7-expressing transgenic trophozoites as per previously established protocol [41 (link)]. Briefly, transgenic cells were lysed by freeze-thawing using liquid nitrogen. Soluble (supernatant) and membrane-enriched fractions (pellet) were isolated using centrifugation at 14,000×g for 10 min at 4 °C. Both fractions were subject to immunoprobing using a rat-derived monoclonal anti-HA antibody (dilution 1:500, Roche) followed by a secondary anti-rat antibody coupled to horseradish peroxidase (dilution 1:2000; Southern Biotech) and Coomassie staining. Non-fractionated whole cell lysates from both non-transgenic and HA-GiCHMP7-expressing parasites were prepared and analysed similarly.

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Pipaliya S.V., Santos R., Salas-Leiva D., Balmer E.A., Wirdnam C.D., Roger A.J., Hehl A.B., Faso C, & Dacks J.B. (2021). Unexpected organellar locations of ESCRT machinery in Giardia intestinalis and complex evolutionary dynamics spanning the transition to parasitism in the lineage Fornicata. BMC Biology, 19, 167.

Publication 2021

rat-derived monoclonal anti-HA antibody

Anti antibody Cell Centrifugation Dilution Fractionation Freeze Horseradish peroxidase Membrane Monoclonal antibody Nitrogen Parasites Transgenic Trophozoites

Corresponding Organization : Czech Academy of Sciences, Biology Centre

Other organizations : University of Alberta, University of Zurich, Dalhousie University, University of Bern

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Variable analysis

independent variables

Expressing HA-GiCHMP7 transgenic trophozoites

dependent variables

Localization and expression of HA-GiCHMP7 protein in soluble (supernatant) and membrane-enriched (pellet) fractions

control variables

Non-transgenic trophozoites

controls

Positive control: HA-GiCHMP7-expressing transgenic trophozoites
Negative control: Non-transgenic trophozoites

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