Rat derived monoclonal anti ha antibody

Chemically fixed cells for subcellular recombinant protein localization were prepared as previously described [118 (link)]. HA-epitope tagged recombinant proteins were detected using a rat-derived monoclonal anti-HA antibody (dilution 1:200, Roche) followed by a secondary anti-rat antibody coupled to AlexaFluor 488 fluorophores (dilution 1:200, Invitrogen). Samples were embedded in Vectashield (VectorLabs) or Prolong Diamond Mounting medium (Invitrogen) containing 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining.

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.