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

Wash one chicken egg with 1% SDS in distilled water, and separate the egg yolk from the egg white under sterile conditions.

Prepare solutions A, B, C, and D. Add 500 microliters of solutions A and C to solution B, and 100 microliters of solution D. Mix them and pour 25 milliliters into each Petri dish. Wait for the solution to solidify under sterile conditions, and make wells of approximately 2 to 3 millimeters in diameter, with a thin tube.

Add a total of 20 microliters of phosphate buffer as a negative control in one well, and 20 microliters of a determined phospholipase as a positive control in another well.

Place different amounts of the crude extract protein - 5, 15, 25, 35, and 45 micrograms in the remaining wells, each in a final volume of 20 microliters. Incubate at 37 degrees Celsius for 20 hours.