LUNDSTROM IDENTIFICATION OF FISH SPECIES 



is measured using a 3-mm diameter Ingold micro- 

 combination surface pH electrode and Corning 

 Model 101 digital pH meter. The electrode was 

 calibrated with standard pH buffer solutions at 

 room temperature. 



The protein patterns were stained with Coo- 

 massie Blue R-250 and destained in \Q'7e ethanol- 

 109? acetic acid (Righetti and Drysdale 1974). 

 After destaining, the gels may be air dried and 

 stored indefinitely. 



RESULTS AND DISCUSSION 



Figure 1 shows typical protein patterns for 

 11 species of commercially important New En- 

 gland fishes. The pH gradient in this gel runs 

 from pH 3.5 at the top (anode) to pH 10.0 at the 

 bottom (cathode). The pattern for each species 

 appeared to be unique and demonstrated resolu- 

 tion not normally attained by conventional 

 electrophoretic techniques. Closely related spe- 

 cies such as cod and haddock or red hake and 

 white hake show similarities in overall patterns, 

 but enough differences are present to permit a 

 positive identification. 



Due to the large number of protein bands re- 

 solved in the pH 3.5-10.0 gradient, many of which 

 have the same isoelectric point, it is sometimes 

 advantageous to look at only a small portion of 

 the pattern under increased resolution. Figure 2 

 shows the same 11 species compared in a pH 3.5- 

 5.0 gradient. The resolution is much greater and 

 identification is not complicated by the presence 

 of as many proteins with the same isoelectric point 

 from species to species. 



Figures 3 and 4 illustrate the reproducibility of 

 the protein patterns through a time interval. The 

 proteins in Figure 3 were focused in 2.5 h using 

 a constant power of 10W. The proteins in Figure 4 

 were focused in 5.5 h using a constant-voltage 

 power supply. The voltage was manually in- 

 creased from 100 V to 300 V in hourly 100-V 

 intervals. The voltage was then held constant at 

 300 V for 3.5 h. The proteins in both plates have 

 been focused to equilibrium, and the pattern for 

 each species is reproducible. 



The protein patterns one obtains in isoelectric 

 focusing are dependent on the pH gradient formed 

 in the gel. Commercially prepared carrier ampho- 

 lytes form pH gradients that remain stable and 

 reproducible during the time necessary for the 

 complete equilibrium focusing of sarcoplasmic 



proteins. Figure 5 shows the pH gradients formed 

 in the previous two figures. The pH gradient curve 

 labeled "A" corresponds to the plate in Figure 3, 

 and the one labeled "B" corresponds to the plate 

 in Figure 4. The slightly lower position of pH 

 gradient A is also seen by the displacement of 

 the patterns in Figure 3 toward the lower end of 

 the gel (cathode). This slight shift of the pH 

 gradient, however, was not enough to affect the 

 reproducibility of the protein patterns. 



Isoelectric focusing offers several advantages 

 over electrophoretic techniques for the identifica- 

 tion offish species. Isoelectric focusing is an equi- 

 librium technique where the proteins are limited 

 in how far they can travel by the pH gradient. 

 Since proteins have a net charge of zero at their 

 isoelectric point, no migration beyond that point 

 can take place. Diffusion of the isoelectric proteins 

 is prevented by the electric field. During the 

 course of a normal electrofocusing experiment, 

 as long as the pH gradient remains stable, the 

 protein patterns will not vary. In contrast, protein 

 patterns from conventional electrophoretic tech- 

 niques are time dependent and may suffer loss 

 of resolution due to diffusion. 



Another advantage of isoelectric focusing over 

 conventional electrophoretic techniques is the 

 ease of sample application. Samples were applied 

 directly from micropipettes into molded sample 

 wells. Samples may also be applied as a drop or 

 streak on the gel surface or by placing a small 

 rectangle of filter paper saturated with the sample 

 directly on the gel. The position of sample appli- 

 cation may be at any point on the gel slab. While 

 some of these sample application techniques may 

 be common to other electrophoretic procedures, 

 only in IEF may these techniques be used inter- 

 changeably without affecting the protein pat- 

 terns. This versatility is an important asset. 

 Dilute extracts (e.g., when the amount of muscle 

 tissue available is unavoidably small) may be 

 applied in a large volume to obtain a protein 

 pattern comparable to that obtained with a small 

 volume of a concentrated extract (e.g., a drip fluid 

 sample from a recently frozen fish). Large sample 

 volumes may also be applied so that minor com- 

 ponents may be detected and compared between 

 species. The ability to vary the position of sample 

 application without affecting the protein pattern 

 eliminates one more possibility for human error. 

 Sample application technique in conventional 

 electrophoretic methods affects the protein pat- 

 tern. Samples must be carefully applied as a thin 



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