FISHERY BULLETIN: VOL. 75, NO. 3 



cester fish processors. Four specimens of each 

 species were examined except for cod and haddock 

 where 15 individuals each were examined. All 

 fish were held on ice from purchase to filleting. 

 Fillets were held at 8°C until extraction of sarco- 

 plasmic proteins. 



Sarcoplasmic protein extracts were prepared by 

 blending 100 g of muscle tissue with 200 ml of 

 distilled water in a 500-ml Waring 2 blender jar. 

 A Teflon baffle shaped to fit the inside contour 

 of the blender jar about 1 cm below the water 

 level was used to prevent the incorporation of 

 air bubbles during the blending operation. The 

 distilled water, blender jar, and baffle were chilled 

 to 8°C prior to use to prevent protein denaturation 

 from heat generated during blending. The result- 

 ing mixture was centrifuged at 1,400 g for 30 min 

 at 4°C in an International PR-2 Refrigerated 

 Centrifuge. The resulting supernatant was used 

 for analysis without any further purification. 



Preparation of Polyacrylamide Gel Slab 



The polyacrylamide gel slab was chemically 

 polymerized between a glass plate and an acrylic 

 template. The glass plate and acrylic template 

 were separated by a 0.75-mm acrylic spacer that 

 extended around three sides leaving the top open. 

 The template had embedded teeth that formed 

 sample wells in the gel surface. The gel slabs used 

 in these experiments were 175 mm x 90 mm x 

 0.75 mm and contained 12 sample wells, each 

 capable of holding up to 5 ju.1. 



A 4% (wt/vol) polyacrylamide gel containing 

 2% (wt/vol) carrier ampholytes was prepared as 

 follows: 



Into a 25-ml Erlenmeyer flask was pipetted 



8.2 ml distilled water 



3.0 ml 50% (vol/vol) glycerol (final concentra- 

 tion 10% [vol/vol]) 



3.0 ml 20% (wt/vol) acrylamide (final concen- 

 tration 4% [wt/vol]) plus 0.8% (wt/vol) 

 bisacrylamide (final concentration 0.16% 

 [wt/vol]) 



5.0 /u.1 tetramethylethylenediamine (final 

 concentration 0.03% [wt/vol | ) 



0.75 ml 40% (wt/vol) ampholine of appro- 

 priate pH range (final concentration 2% 

 I wt/vol |). 



2 Reference to trade names does not imply endorsement by the 

 National Marine Fisheries Service, NOAA. 



This solution was degassed under vacuum for 

 4 min. Polymerization was started with the addi- 

 tion of 50 fx\ 10% (wt/vol) ammonium persulfate 

 (final concentration 0.03% [wt/vol]). After a final 

 degassing under vacuum for one more minute, 

 the solution was immediately pipetted into the 

 gel mold. The top of the gel solution was layered 

 with water to form an even surface. Polymeriza- 

 tion was complete in 20 min at room temperature. 

 The open top of the gel mold was then sealed with 

 masking tape, and the whole assembly was placed 

 in a refrigerator (8°C) overnight before use. A 

 supply of gel slabs may be prepared and stored 

 for 2 wk in this manner. After the gel had polymer- 

 ized, the template and spacer were removed leav- 

 ing the gel adhering to the glass plate. 



Electrofocusing Procedure 



Electrofocusing was carried out using a Medical 

 Research Apparatus Slab Electrofocusing Appa- 

 ratus, Model M-150. The gel slab was placed on 

 the cooling platform and cooled to -2°C prior to 

 sample application. To insure good thermal con- 

 tact, a layer of light paraffin oil was used between 

 the glass plate and the cooling platform. After 

 the gel slab had cooled, 5 /x\ of the protein extract 

 was pipetted into a sample well with a micro- 

 pipette. Up to 12 samples may be compared in a 

 single gel slab. Felt strips soaked in 1M NaOH 

 (catholyte) and 1M H 3 P0 4 (anolyte) were applied 

 to the edges of the gel to provide electrical contact 

 with the platinum wire electrodes. A power supply 

 was connected to the electrodes, and power was 

 applied until equilibrium focusing was attained. 

 Both constant-power and constant-voltage power 

 supplies were used in these experiments. In iso- 

 electric focusing, a power supply capable of 

 delivering constant power is preferred. Using a 

 constant power of 10 W, equilibrium focusing 

 was complete in 1.5-2.0 h. Using constant 

 voltage, the voltage must be manually increased 

 to compensate for increased resistance through 

 the gel as the pH gradient forms. Separation times 

 are longer (5-6 h) and resolution suffers due to 

 joule heating within the gel. With either type of 

 power supply, equilibrium focusing is attained 

 and the reproducibility of the protein patterns 

 is not affected. After electrofocusing is complete, 

 the pH gradient may be measured as a check on 

 reproducibility or to determine the isoelectric 

 points of the separated proteins. The plate is 

 warmed to room temperature and the pH gradient 



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