Lipid Binding Assays for Defensins

Lipid-binding assays were performed using PIP StripsTM or SphingoStripsTM (lipid–protein interaction assay, Echelon Biosciences, Salt Lake City, UT, USA) as described in [9 (link),22 (link)]. Ppdef1’s lipid binding was detected using polyclonal antibodies generated in rabbits specific to the Ppdef1, DmAMP1 or NaD1 defensins using Gel Doc (Biorad). The PIP Strips are hydrophobic membranes with 100 pmol of the following lipids: Lysophosphatidic acid (LPA), Lysophosphocholine (LPC), Phosphatidylinositol (PI), Phosphatidylinositol (3)-phosphate (PI(3)P), Phosphatidylinositol (4)-phosphate (PI(4)P), Phosphatidylinositol (5)-phosphate (PI(5)P), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Sphingosine 1-Phosphate (S1P), Phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P₂), Phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P₂), Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂), Phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃), Phosphatidic acid (PA) and Phosphatidylserine (PS). SpingoStrips are hydrophobic membranes with 100 pmol of the following lipids: Sphingosine (S), Sphingosine 1-phosphate (S1P), Phytosphingosine (PhS), Ceramide (Cer), Sphingomyelin (SM), Sphingosylphosphorylcholine (SPC), Lysophosphatidic Acid (LPA), Myriosine (Myr), Monosialoganglioside-GM1 (GM1), Disialoganglioside-GD3 (GD3), Sulfatide, Psychosine, Cholesterol (C), Lysophosphatidylcholine (LPC) and Phosphatidylcholine (PC). A 5 µL volume of defensin (5 µg/mL) was spotted onto the membrane as a positive control and allowed to dry before blocking.

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Parisi K., McKenna J.A., Lowe R., Harris K.S., Shafee T., Guarino R., Lee E., van der Weerden N.L., Bleackley M.R, & Anderson M.A. (2024). Hyperpolarisation of Mitochondrial Membranes Is a Critical Component of the Antifungal Mechanism of the Plant Defensin, Ppdef1. Journal of Fungi, 10(1), 54.

Publication 2024

Corresponding Organization : Hexima (Australia)

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Variable analysis

independent variables

Lipid-binding assays were performed using PIP StripsTM or SphingoStripsTM

dependent variables

Ppdef1's lipid binding was detected using polyclonal antibodies generated in rabbits specific to the Ppdef1, DmAMP1 or NaD1 defensins

control variables

A 5 µL volume of defensin (5 µg/mL) was spotted onto the membrane as a positive control and allowed to dry before blocking

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