Western Blot Analysis of Parasite Proteins

Protein samples were separated using NuPAGE Bis/Tris gels, as described [15 (link)], loading 2.5 × 10⁶ parasite equivalents per lane. Membranes were probed with rat anti-HA antibodies (1:100 to 1:3,000 dilutions; clone 3F10, Sigma, 11867423001), rabbit anti-TgTom40 [54 (link)] (1:2,000 dilution), mouse anti-GFP (1:1,000 dilution; Sigma, 11814460001), mouse anti-BAG1 [55 (link)] (1:250 dilution; a kind gift from Louis Weiss, Albert Einstein College of Medicine), rabbit anti-SAG1 (1:1,000 dilution; a kind gift from Michael Panas and John Boothroyd, Stanford University), or mouse anti-TgGRA8 [56 (link)] (1:100,000 dilution; a kind gift from Gary Ward, U. Vermont) as primary antibodies, and horseradish peroxidase-conjugated goat anti-rat (1:5,000 to 1:10,000 dilutions; Santa Cruz, sc-2006, or Abcam, ab97057), goat anti-rabbit (1:5,000 to 1:10,000 dilution; Santa Cruz, sc-2004, or Abcam, ab97051), or goat anti-mouse (1:5,000 to 1:10,000 dilution; Santa Cruz, sc-2005) secondary antibodies.

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Rajendran E., Clark M., Goulart C., Steinhöfel B., Tjhin E.T., Gross S., Smith N.C., Kirk K, & van Dooren G.G. (2021). Substrate-mediated regulation of the arginine transporter of Toxoplasma gondii. PLoS Pathogens, 17(8), e1009816.

Publication 2021

clone 3F10 mouse anti-GFP horseradish peroxidase-conjugated goat anti-rat ab97057 ab97051

Anti antibodies Antibodies Bag1 Bis tris Clone Dilution Gels Goat Horseradish peroxidase Medicine Membranes Mouse Parasite Protein Rabbit

Corresponding Organization : University of Technology Sydney

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Variable analysis

independent variables

Protein samples were separated using NuPAGE Bis/Tris gels
Parasite equivalents loaded per lane (2.5 × 10^6)

dependent variables

Protein expression levels detected by Western blot using various antibodies

control variables

Protein samples were separated using NuPAGE Bis/Tris gels, as described [15 (link)]
Antibody dilutions used for Western blot: rat anti-HA (1:100 to 1:3,000), rabbit anti-TgTom40 (1:2,000), mouse anti-GFP (1:1,000), mouse anti-BAG1 (1:250), rabbit anti-SAG1 (1:1,000), mouse anti-TgGRA8 (1:100,000)
Secondary antibodies used for Western blot: horseradish peroxidase-conjugated goat anti-rat (1:5,000 to 1:10,000), goat anti-rabbit (1:5,000 to 1:10,000), or goat anti-mouse (1:5,000 to 1:10,000)

positive controls

Rabbit anti-TgTom40 [54 (link)]
Mouse anti-BAG1 [55 (link)]
Rabbit anti-SAG1
Mouse anti-TgGRA8 [56 (link)]

negative controls

Not explicitly mentioned

Annotations

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