GB/T 18654.13-2026Inspection of germplasm for fishes - Part 13: Test method for isozyme electrophoresis (English PDF)

5.1 Reagents

5.1.1 Glacial acetic acid (CH3COOH).

5.1.2 Ethanol (CH3CH2OH).

5.1.3 Glycerol (C3H8O3).

5.1.4 Sodium hydroxide (NaOH).

5.1.5 Nitrotetrazolium chloride (NBT). Store at 4°C.

5.1.6 Tris(hydroxymethyl)aminomethane (Tris).

5.1.7 Citric acid (C6H8O7).

5.1.8 Ethylenediaminetetraacetic acid (EDTA).

5.1.9 Boric acid (H3BO3).

5.1.10 L-histidine (C6H9N3O2).

5.1.11 Acrylamide (Arc).

5.1.12 N,N'-methylenebisacrylamide or methylenebisacrylamide (Bis).

5.1.13 Tetramethylethylenediamine (TEMED).

5.1.14 Ammonium persulfate (AP).

5.2 Solution Preparation 5.2.1 2.5% glacial acetic acid. Measure

2.5 mL of glacial acetic acid (5.1.1), add

97.5 mL of water, and mix well.

5.2.2 Fixative. 3 parts ethanol (5.1.2), 1 part glacial acetic acid (5.1.1), 1 part glycerol (5.1.3), and 5 parts water, mix well. 5.2.3 5% sodium hydroxide solution. Weigh 5g of sodium hydroxide (5.1.4) and dilute to 100mL with water. 5.2.4 1 mg/mL Nitrotetrazole Blue Chloride Solution. Weigh 100 mg of nitrotetrazole blue chloride (5.1.5) and dilute to the final volume with water. 100mL.

5.2.5 TC gel-forming buffer. Weigh 3.0285 g of tris(hydroxymethyl)aminomethane (5.1.6), adjust the pH to

8.0 with citric acid (5.1.7), and add water. Bring the volume to 1L.

5.2.6 EBT gel preparation buffer. Weigh 5.4513 g of tris(hydroxymethyl)aminomethane (5.1.6) and 0.2922 g of ethylenediaminetetraacetic acid (5.1.8), and use... Adjust the pH to

8.6 with boric acid (5.1.9) and bring the volume to 1L with water.

5.2.7 HC gelation buffer. Weigh 1.9395g histidine (5.1.10) and 21.014g citric acid (5.1.7), and use 5% sodium hydroxide solution. (5.2.3) Adjust the pH to

8.2 and bring the volume to 1L with water.

5.2.8 TC Electrode Buffer. Weigh 4.865g of tris(hydroxymethyl)aminomethane (5.1.6), adjust the pH to

8.0 with citric acid (5.1.7), and dilute with water. Fill to 1L.

5.2.9 EBT Electrode Buffer. Weigh 65.4g of tris(hydroxymethyl)aminomethane (5.1.6) and 3.51g of ethylenediaminetetraacetic acid (5.1.8), and use boric acid... (5.1.9) Adjust the pH to

8.6 and bring the volume to 15L with water.

5.2.10 HC Electrode Buffer. Weigh 1.9395g L-histidine (5.1.10) and 21.014g citric acid (5.1.7), and dissolve in 5% sodium hydroxide. Adjust the pH of solution (5.2.2) to 8.5, and then bring the volume to 10L with water.

5.2.19 Electrode buffer stock solution. Weigh

6.00 g of tris(hydroxymethyl)aminomethane (5.1.6) and

28.8 g of glycine (5.1.18), and use hydrochloric acid... (5.1.15) Adjust to pH 8.3, then bring the volume to 1L with water. Adjust as needed.

5.2.20 Gel reservoir (Arc.Bis solution). Weigh 93.75g acrylamide (5.1.11), 2.5g N,N'-methylenebisacrylamide or methyl methacrylate (MCMA). Add bisacrylamide (5.1.12) and dilute to 250 mL with water. 5.2.21 1750 mg/mL ammonium persulfate solution (prepared fresh). Weigh

17.5 g of ammonium persulfate (5.1.14) and dissolve it in 10 mL of water.

5.2.22 Staining solution. Several common isoenzyme staining solutions are prepared according to Appendix A.

5.3 Materials

5.3.1 Syringe. 5mL.

5.3.2 Centrifuge tubes. 1.5mL in size.

5.3.3 Glass homogenizer.

5.3.4 Glue making mold.

5.3.5 Micro-volume syringe. volume range 1µL~20µL, accuracy 0.3%~1.5%.

5.3.6 Dyeing vat.

7.2 Sample Preparation

7.2.1 Serum Samples Use a syringe (5.3.1) to draw at least 300 µL of blood from the caudal artery of a live fish, transfer it into a centrifuge tube (5.3.2), and centrifuge at 4°C in a high-speed refrigerated centrifuge. In (6.4), the serum was separated by centrifugation at 3500 r/min for 1 min and stored separately at 4°C for later use.

7.2.2 Tissue Samples Vivisection was performed, and appropriate amounts of tissue were taken, numbered, and placed into centrifuge tubes (5.3.2). These tubes were then stored in a -18°C freezer (6.5) for later use. Use. Take 0.1g~0.3g of sample, add 3 times the volume of NAD (5.1.22) to one part of the sample, and homogenize in a micro homogenizer (6.6) with... Homogenize at 4000 rpm for 2 minutes, or homogenize using a glass homogenizer (5.3.3) under ice bath conditions. Transfer the homogenized sample into centrifuge tubes. (5.3.2) Centrifuge at 15000 r/min for 10 min in a high-speed refrigerated centrifuge (6.4) at 4°C, and collect the supernatant. Store separately at 4°C. spare.

8.1 Glue Preparation

8.1.1 Preparation of horizontal electrophoresis gels Preparation method of 4% polyacrylamide gel. Measure

13.2 mL of L Arc.Bis solution (5.2.11),

14.7 mL of gel preparation buffer (

5.2.6 or 5.2.7),

0.3 mL 25% tetramethylethylenediamine (5.2.12),

0.6 mL 100 mg/mL ammonium persulfate solution (5.2.13) After mixing, immediately pour the mixture into the gel mold (5.3.4). After the polymerization reaction is complete, remove the gel for later use.

8.1.2 Preparation of Vertical Electrophoresis Gel The preparation method for discontinuous gels (7.5% separating gel and 3.5% stacking gel) is shown in Table 1, and can be adjusted appropriately as needed. Use a gel-making mold. In (5.3.4), first pour in the separating gel. After the separating gel coagulates, pour in the concentrating gel and insert the comb. After the concentrating gel polymerizes, remove the comb for later use. Table

1.Preparation of 7.5% separating gel and 3.5% stacking gel 7.5% separating gel, 3.5% stacking gel Separating gel buffer (5.2.17)

1.2 mL; Stacking gel buffer (5.2.18)

1.25 mL Gel reservoir (5.2.20) 7mL Gel reservoir (5.2.20) 1mL

0.3 mL of 1750 mg/mL ammonium persulfate solution (5.2.21)

0.2 mL of 1750 mg/mL ammonium persulfate solution (5.2.21) 25% Tetramethylethylenediamine (5.2.12) 50 µL 25% Tetramethylethylenediamine (5.2.12) 40 µL

21.45 mL of water

7.51 mL of water Total volume 30mL Total volume 10mL

8.2 Add buffer solution

8.2.1 Horizontal electrophoresis tank Before the experiment, 4% polyacrylamide gel (8.1.1) was placed on a cooling plate with paper bridges on both sides, and then added to the left and right electrophoresis tanks (6.8). The appropriate electrode buffer solution (5.2.8, 5.2.9, or 5.2.10). Cool to 4°C.

8.2.2 Vertical Electrophoresis Tank Before the experiment, the discontinuous gel (8.1.2) was fixed in the vertical electrophoresis tank (6.8), and electrode buffer was added to both the upper and lower electrophoresis tanks (6.8). (

5.2.9 or 5.2.10). Cool to 4°C.

8.3 Spotting Add 10 µL of sample (

7.2.1 or 7.2.2) to the gel spot well using a microsyringe (5.3.5), adjusting as needed.

8.4.1 Horizontal Electrophoresis

8.4.1.1 Pre-electrophoresis. Electrophoresis at 25mA for 10 minutes. Adjustments may be made as needed.

8.4.1.2 Formal electrophoresis. Constant voltage electrophoresis or constant current electrophoresis is performed under appropriate voltage or current. The electrophoresis time depends on the type of isoenzyme.

8.4.2 Vertical electrophoresis

8.4.2.1 Pre-electrophoresis. Electrophoresis at 25mA for 30 minutes. Adjustments may be made as needed.

8.4.2.2 Formal electrophoresis. Constant voltage electrophoresis or constant current electrophoresis is performed under appropriate voltage or current. The electrophoresis time depends on the type of isoenzyme.

8.5 Staining After electrophoresis, place the gel in the staining solution corresponding to the enzyme to be detected (Appendix A) and stain at room temperature in the dark. When all bands are visible... Stop staining once the stain is clear.

8.6 Decolorization The stained gel was placed in 2.5% glacial acetic acid (5.2.1) for decolorization until the gel background was clear and transparent.

8.7 Fixed After decolorization, the electrophoresis gel is placed in the fixative (5.2.2) and fixed for at least half an hour.

8.8 Take a photo The isozyme bands in the gel were photographed and saved using a photographic device (6.10).