NY/T 1663-2008Determination of ��-lactoglobulin content in milk and dairy products SDS-PAGE electrophoresi (English PDF)

4.1 Glacial acetic acid (CH3COOH)

4.20 Running gel buffer stock solution, 1.5 mol/L, pH 8.8

Accurately take 9.08g of tris(hydroxymethyl)aminomethane (4.9); dissolve it in 40 mL of water; adjust the pH to 8.8 by hydrochloric acid solution (4.17); use water to make the constant volume to 50mL; and store at 4°C.

4.21 Persulfate solution, 100g/L

Accurately take 0.1g of ammonium persulfate (4.4); add 1mL of water to dissolve; and prepare before use.

4.22 Dodecyl sulfonic acid sodium solution, 100g/L

Accurately take 5g of dodecyl sulfonic acid sodium (4.13); dissolve in water and make constant volume to 50mL; and store at room temperature.

4.23 N, N, N ', N'-Tetramethylethylenediamine solution with volume fraction of 10%

Take 0.10mL of N, N, N ', N'-Tetramethylethylenediamine (4.14); dilute with water and make constant volume to 1.00 mL.

4.24 Sample buffer, 0.08mol/L

Accurately take 4mg of bromophenol blue (4.15); dissolve it in 5.00mL water.

Respectively, measure 1.60mL of stacking gel buffer stock solution (4.19), 4.00mL of dodecyl sulfonic acid sodium solution (4.22), 1.20mL of beta-mercaptoethanol (4.10),

2.20mL of glycerin (4.7); all mixed, then dilute with water and make constant volume to 20mL; and stored at 4°C.

4.25 Electrode buffer, pH 8.3

Accurately take 3.0g of tris(hydroxymethyl)aminomethane (4.9) and 14.4g of glycine (4.8); add 10 mL of dodecyl sulfonic acid sodium solution (4.22); and adjust the pH to

8.3; make constant volume to 1000mL.

4.26 Coomassie brilliant blue staining solution, 2.5g/L

Take 0.25g of Coomassie brilliant blue R-250 (4.16) and 10g of ammonium sulfate (4.3); respectively, add 20mL of ethanol (4.6) and 10mL of phosphoric acid (4.2);

dissolve and mix; and use water to make constant volume of 100mL.

4.27 De-staining solution

Respectively, take 75mL of glacial acetic acid (4.1), 50mL of methanol (4.5) and 875mL of water; and mix evenly.

4.28 beta-lactoglobulin standard solution, 1.00 mg/mL

Accurately take 0.0100g of beta-lactoglobulin standard substance (purity >=90%); use the sample buffer (4.24) to make constant volume to 10mL; heat in a boiling water bath for

3min~5min; and store at -20°C below.

6.1 Specimen preparation

6.1.1 Liquid sample

Take 1mL of sample; sequentially add 1mL of water and 2mL of sample buffer (4.24);

heat in boiling water bath for 3min~5min; stir by magnetic-stirrer (5.7) for 4h; centrifuge for 5min; remove fat; take supernatant and sub-package; store at -20°C for later-use.

6.1.2 Solid sample

Take 1g of sample, accurate to 0.1 mg; add appropriate amount of water to dissolve;

make constant volume to 10mL; and operate according to 6.1.1.

6.2 Preparation of running gel

Prepare 20mL of 12% running gel according to Table 1; mix it and then add it to the gap between the long and short glass plates, which is about 60mm~70mm high. Slowly inject water about 5mm high along the long glass plate wall; and seal the water. After about 30min, the boundary between the gel and the water seal layer with different

6.8 De-staining

After staining the gel, first rinse its surface to remove the excessive stain; and then soak it into the de-staining solution (4.27) for de-staining. Change the de-staining solution until the gel background is colorless.

6.9 Analysis

Use the densitometer to measure and analyze the gel, and calculate the content of beta- lactoglobulin according to the optical density value. For a typical map, see Appendix A.