IDENTIFICATION OF FISH SPECIES BY THIN-LAYER 

 POLYACRYLAMIDE GEL ISOELECTRIC FOCUSING 



Ronald C. Lundstrom* 



ABSTRACT 



Conventional electrophoretic techniques for the identification offish species are limited in the resolu- 

 tion 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 (IEF), 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 ipH 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 offish 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 (Connell 1953). 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 electro- 

 phoresis was applied as a method for diffentiating 

 fish species (Thomson 1960). Subsequent attempts 

 to further improve species identification tech- 

 niques centered on the investigation of new sta- 

 bilizing 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 effective- 

 ness as a routine species identification test. Varia- 

 tions 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 identi- 

 fication procedures, disc electrophoresis (Thomson 

 1967) and cellulose acetate electrophoresis (Lear- 

 son 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, thin- 

 layer 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 of zero, 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. 



PROCEDURE 



Isolation of Sarcoplasmic Proteins 



Fresh iced fish was obtained from various Glou- 



571 



