Identification of Fish Species by Thin-layer Polyacrylamide Gel Isoelectric Focusing

Conventional electrophoretic techniques for the identification offish species are limited in the resolution and reproducibility needed for the reliable identification of fish species. This paper describes the potential of a high resolution protein separation technique, thin-layer polyacrylamide gel isoelectric focusing (IEFl, as a new means of identifying fish species. Sarcoplasmic protein patterns are shown for 11 species of commercially important New England fishes under both low resolution (pH 3.5-10 gradient) and high resolution (pH 3.5-5 gradient) conditions. The reproducibility of the protein patterns and pH gradients from day to day is also shown. The inherent high resolution and excellent reproducibility of IEF should allow the positive identification of fish species without the costly procedure of maintaining a supply of known species for use as standards. Many different electrophoretic techniques have been used for the identification of fish species. Protein extracts from several species of fishes were first compared using moving boundary electrophoresis (Conne111953). Differences in the electrophoretic protein patterns between species formed a "fingerprint" for each. In an effort to obtain higher resolution and reproducibility of the protein patterns, starch gel zone electrophoresis was applied as a method for diffentiating fish species (Thomson 1960). Subsequent attempts to further improve species identification techniques centered on the investigation of new stabilizing media. The use of polyacrylamide gels (Payne 1963; Cowie 1968) and agar gels (Hill et al. 1966) resulted in shortened analysis times, increased resolution, and easier handling and storage of gels. A rapid identification technique based on cellulose acetate electrophoresis (Lane et al. 1966) has found widespread use in quality control. Each of these electrophoretic techniques (except moving boundary electrophoresis) is still in common use and has contributed much towards eliminating problems of species substitution. Unfortunately, each of these techniques is subject to one or more limitations that lessen its effectiveness as a routine species identification test. Variations in stabilizing media composition, sample application technique, separation time, applied !Northeast Fisheries Center Gloucester Laboratory, National Marine Fisheries Service, NOAA, Emerson Avenue, Gloucester, MA 01930. Manuscript accepted February 1977. FISHERY BULLETIN: VOL. 75, NO.3, 1977. voltage or current, and the technician's skill indicated the need for simultaneously running known species along with unknown samples to obtain a reliable identification. Collaborative studies of the two most widely used species identification procedures, disc electrophoresis (Thomson 1967) and cellulose acetate electrophoresis (Learson 1969, 1970), showed that reproducibility of specific protein patterns from analysis to analysis was a major problem. This paper describes the potential of a high resolution protein separation technique, thinlayer polyacrylamide gel isoelectric focusing (IEF), as a new means of identifying fish species. IEF is an equilibrium technique in which proteins are separated according to their isoelectric points in a reproducible natural pH gradient. The pH gradient is formed in the gel by the electrolysis of amphoteric buffer substances called carrier ampholytes. Protein molecules migrate in the electric field along the pH gradient until they reach the pH equal to their isoelectric point. Here the protein has a net charge ofzero, and no further migration can take place. The proteins become concentrated into very sharp bands and molecules whose isoelectric points differ by 0.07 pH units (pH 3.5-10 gradient) or 0.02 pH units (pH 3.5-5 gradient) may be resolved.