To compare the quality of extracted proteins with pro- teins obtained by other venom extraction methods, we prepared a bidimensional gel electrophoresis (2DE gel) using about 300 mg of putative protein from an aqueous phase extraction from S. invicta, and a 2DE gel of pure venom protein extract purchased from Vespa Labs Inc. (Spring Mills, PA, USA) (Fig. 2; also refer to Pinto et al., 2012). Gels were digitalized with a table scanner, and the software Adobe Photoshop CS was used to discard color information, normalize contrast between images, and number the obtained spots. The general patterns of the two 2DE gels are clearly similar. Indeed, most proteins are found at similar isoelectric points vs. molecular size positions, and the number of obtained proteins was almost identical. Furthermore, preliminary results of mass spectrometry analysis of individual protein spots in similar positions showed no difference in protein content; the individual proteomic identifications of fire ant venom are dealt within a separate publication (Pinto et al., 2012).
The described method of venom extraction is rapid and inexpensive, and depends only on the ability of locating and handling fire ants and the necessary solvents. This method can likely be adapted for venom extraction from other aggressive hymenopterans (e.g., other ants, or cold- anesthetized bees and wasps). Furthermore, the protocol may be further revisited and optimized to increase the purity of each fraction and possibly replace the used sol- vents with environment-friendly alternatives (e.g., using ethanol or cold acetone). We hope that the presented method will encourage investigators to advance the study of venom proteins and peptides of fire ants and other venomous insects.