Phospholipase Activity Assay: A Gel Diffusion Assay to Determine Phospholipase Activity in Crude Venom Extract from Anthopleura dowii

Published: April 30, 2023

Abstract

Source: Ramírez-Carreto, S., et al., Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays. J. Vis. Exp. (2022)

In this video, we demonstrate an assay to establish the presence of phospholipase in the crude venom extract from Anthopleura dowii—a marine sea anemone. The assay utilizes an agarose gel diffusion technique, in which the enzyme phospholipase diffuses and breaks down phospholipids incorporated in the solid media in a radial manner.

Protocol

1. Organism collection Collect sea anemone A. dowii. NOTE: Here, the organisms were collected during low tide periods in the intertidal zone off the coast of Ensenada Baja, California, Mexico (Figure 1A, B).  Transport the organisms to the laboratory in containers with seawater (Figure 1C). In the laboratory, clean the organisms with distilled water by hand to remove the big substrate particles adhering to the body.</l…

Representative Results

Figure 1: Sea anemone collection. (A) Intertidal zone in El Sauzal, Baja California Norte, Mexico. (B) Sea anemone collected. (C) Anthopleura dowii Verrill, 1869. Table 1: Quantitation of protein by Bradford assay. …

Divulgazioni

The authors have nothing to disclose.

Materials

15 mL conical centrifuge tube Corning 430766
2-Bromophenol blue Sigma B75808
2-mercaptoetanol Sigma-Aldrich M6250-100ML
50 mL conical centrifuge tubes Corning 430828
Acetic Acid Glacial J.T. Baker 9515-03
Acrylamide Promega V3115
Agarose Promega V3125
Bisacrylamide Promega V3143
Bovine Serum Albumin Fraction V Sigma A3059-100G
Bradford Protein Assays Bio-Rad 5000006
Calcium chloride Sigma-Aldrich C3306
Cell culture plates 96 well, V-bottom Corning 3894
Centrifuge Eppendorf 5804R
Centrifuge tubes Corning CLS430829
ChemiDoc MP system Bio-Rad 1708280
Citric acid Sigma-Aldrich 251275
Clear flat.bottom 96-Well Plates Thermo Scientific 3855
Coomassie Brilliant Blue G-250 Bio-Rad #1610406
Coomassie brilliant blue R-250 Bio-Rad 1610400
Dextrose J.T. Baker 1916-01
Ductless Enclosure Labconco Vertical https://imagej.nih.gov/ij ImageJ 1.53c
Gel Doc EZ Bio Rad. Gel Documentation System
Glycerol Sigma-Aldrich G5516-4L
Hemocytometer Marienfeld 650030
ImageJ (Software) NIH, USA Version 1.53c
Incubator 211 Labnet I5211 DS
Methanol J.T. Baker 9049-03
Mini-PROTEAN tetra cell Bio-Rad 1658000EDU
Na2HPO4 J.T. Baker 3824-01
NaCl J.T. Baker 3624-01
NaH2PO4.H2O J.T. Baker 3818-05
Origin software version 9 To design the plot with sigmoidal adjustments
Petridish Falcon 351007
Pipetman kit Gilson F167380
Precast mini gel BioRad 1658004
Prestained Protein Ladder Thermo Scientific 26620
Protease Inhibitor Cocktail Roche 11836153001
Protein Assay Dye Reagent Concentrate Bio-Rad 5000006
Rhodamine 6G Sigma-Aldrich 252433
SDS Sigma-Aldrich L4509
Sodium citrate dihydrate JT Baker 3646-01
Spectrophotometer THERMO SCIENTIFIC G10S UV-VIS
Tris Base Sigma-Aldrich 77-86-1
Volt Power Supply Hoefer PS300B

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Citazione di questo articolo
Phospholipase Activity Assay: A Gel Diffusion Assay to Determine Phospholipase Activity in Crude Venom Extract from Anthopleura dowii . J. Vis. Exp. (Pending Publication), e21152, doi: (2023).

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