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

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

1. 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).
2. Transport the organisms to the laboratory in containers with seawater (Figure 1C).
3. In the laboratory, clean the organisms with distilled water by hand to remove the big substrate particles adhering to the body.
4. Immediately freeze the organisms at -80 °C in an ultra-freezer for 72 h.
5. Place the organisms in special glasses for freeze-drying, w....

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Na<sub>2</sub>HPO<sub>4</sub>	J.T. Baker	3824-01
NaCl	J.T. Baker	3624-01
NaH<sub>2</sub>PO<sub>4</sub>.H<sub>2</sub>O	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