Amplex red phospholipase d assay kit

Cells were plated, assayed and harvested in 96-well plates. Lysates were prepared using 20 μl/well 0.1% (v/v) Triton X-100 in assay buffer, followed by three freeze-thaw cycles. Lysate was diluted in a further 30 μl of assay buffer before 1:1 addition of assay reagents as per the Amplex Red Phospholipase D Assay Kit (Invitrogen) product protocol.

PLD activity was measured using the Amplex Red Phospholipase D Assay Kit (Invitrogen) according to the manufacturer's instructions. Cell lysates were made of six-well plates using the assay reaction buffer with 1% Triton X-100. For each replicate, 20 μg of lysate was diluted to 50 μl of lysis buffer and added to 50 μl of reaction mixture. Four technical replicates per sample were distributed in a black half-area 96-well plate (Corning). The plate was covered and the reaction proceeded for 30 min at 37 °C before fluorescence was measured.

Precision Plus Protein™ All Blue Standards, BCA reagents, and nitrocellulose membranes were purchased from Bio-RAD (Hercules, CA, USA). EnzChek^® Direct Phospolipase C Assay Kit and Amplex^® Red Phospholipase D Assay Kit were purchased from Invitrogen/Life Technologies (Carlsbad, CA). Chemicals, proteases, protease inhibitors, and antibodies used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted.

Phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) activity assays were performed using manufacturers’ instructions provided with the EnzChek^® Direct Phospolipase C Assay Kit and Amplex^® Red Phospholipase D Assay Kit by Invitrogen/Life Technologies (Carlsbad, CA), respectively. Fluorescence intensity for each assay was measured using a SPECTRAFluor Plus instrument and quantified using associated Magellan^™ software by TECAN over a period of 24 h with the kinetic peaks of the positive controls used for comparison.

The assay was performed using the commercial Amplex Red Phospholipase D Assay Kit (Invitrogen, A12219) to analyze the enzyme activity of PldA in vitro. The assay was carried out at 37 °C for 10 min containing 0.2 µg of PldA, 125 µM di8:0 PC (dissolved in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl) and the supplied regents of the kit. The fluorescence was excited at 530 nm and detected at 590 nm. For the assay of PldA inhibition by PA3488 and VgrG4bCt (aa 693–808), The molar ratio of the samples are: PldA: PA3488 = 1:1, PldA: VgrG4bCt = 1:1, and PldA: PA3488: VgrG4bCt = 1:1:1, repectively. Kinetic study of PldA^FL and PldA^truncate activity were performed under standard assay conditions with various substrate concentrations, ranging from 1 to 200 μM di8:0 PC.