The electrophoretic technique with cellulose acetate membrane was applied to rockfish serum to analyze the change in the amount of serum protein and its fractions which occurred during the course of storage.
The difference of electrophoretic patterns with sex was also examined.
Sampling of the blood and measurements of serum protein, haematocrit, urea nitrogen level of serum were done following the same techniques as used in the previous investigations
5, 6). In addition, the electrophoresis was also used. Immediately after the sampling, the blood was centrifuged for 30 minutes at 2, 500-3, 000rpm to obtain the serum.
The serum was then stored in a refrigerator with temperature between 4° and 7°C.
Electrophoretic apparatus: Separax Micro Analysis (Jo Ko Sangyo Co., Ltd.) with Separax cellulose acetate membrane.
Application of serum: Materials (0.0008-0.001m
l per 1cm wide) were spread 1.5cm from the edge of anode end of a strip.
Electrification: Constant current of 0.8mA/cm width of the strip was applied for 50 minutes. Electric pressure in this case ranged from 145 to 255V.
Buffer solution: Veronal buffer pH 8.6, ionic strength 0.07.
Staining: Ponceau 3R for 90 seconds.
Destaining: 1% glacial acetic acid solution.
Quantitative measurement: After being made transparent with liquid paraffin, electrophero-grams were estimated for extinction at the center with 0.5×0.6mm slit at 500mμ using Atago Self Recording Densitometer (Ozumor Types 7 and 8, Atago Optical Works Co., Ltd.)
To compare the electrophoretic patterns obtained, normal human serum (Moni-Trol 1; Dade Reagents Inc.) was used. The amount of serum protein appeared to increase with the duration of storage (Table 2).
Five peaks were observed clearly in the fresh materials of both sexes just after the sampling of blood. The differentiation of the peaks, however, lessened with the prolongation of preservation. The position of each component, especially of the first peak, showed a trend to move toward the anode (Fig. 1). The electrophoretic patterns seemed reproducible during only 3 days after the sampling (Fig. 1).
The pattern of serum proteins had five components for both male and female. No component equivalent in migration to albumin of normal human serum was observed. The maximum peak for the male appeared at the position of human globulin alpha 2, and that for the female occurred at human globulin beta (Fig. 2). Component-IV was the largest in both male and female (Table 4).
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