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).