A method for detecting α-glucosidase activity
A technology of glucosidase and activity, applied in the field of biological analysis and detection, can solve the problems of measurement error, complex reaction principle, cumbersome experimental operation steps, etc., and achieve the effect of accurate detection
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Embodiment 1
[0074] S1. Weigh 5 mg of sucrose and dissolve it in 1 ml of PBS buffer solution with pH 8 at room temperature;
[0075] S2. Weigh 0.1 mg of α-glucosidase from thermophilic archaea and dissolve it in 1 ml of PBS buffer solution with pH 8 to obtain α-glucosidase solution;
[0076] S3. Measure 1 ml of the sucrose solution in step S1 into the α-glucosidase solution described in step S2, the volume ratio of the two solutions is 1:1, stir at 10 rpm, and react in a water bath at 40°C for 1 minute;
[0077] S4. Place the solution obtained in step S3 into boiling water and boil for 10 minutes, cool to 20°C to inactivate α-glucosidase, and freeze and centrifuge at high speed (-20°C, 50,000 rpm, 5 minutes);
[0078] S5. Centrifuge the supernatant obtained in step S4, take 0.1 ml, put it into a glass capillary (inner diameter 1.5 mm, length 5 cm), and seal at both ends;
[0079] S6. Pack the sample capillary tube obtained in step S5 into the nuclear magnetic test tube containing the TMS ...
Embodiment 2
[0084] S1. Weigh 200 mg of maltoheptaose and dissolve it in 5 ml of citric acid-sodium citrate buffer solution with pH 4.4 at room temperature;
[0085] S2. Weigh 1 mg of α-glucosidase derived from Aspergillus basilica, and dissolve it in 2 ml of citric acid-sodium citrate buffer buffer solution with pH 4.4 to obtain α-glucosidase solution;
[0086] S3. Measure 1 ml of the maltoheptaose solution in step S1 into the α-glucosidase solution described in step S2, the volume ratio of the two solutions is 1:1, stir at 200 rpm, and react in a water bath at 37°C for 20 minutes;
[0087] S4. Place the solution obtained in step S3 in boiling water and boil for 1 minute, cool to 32°C to inactivate α-glucosidase, and freeze and centrifuge at high speed (-4°C, 20,000 rpm, 30 minutes);
[0088] S5. Centrifuge the supernatant obtained in step S4, take 0.5 milliliters, put it into a glass capillary (inner diameter 0.3 millimeters, length 10 centimeters), and seal at both ends;
[0089] S6. T...
Embodiment 3
[0094] S1. Weigh 450mg of maltotetraose, and dissolve it in 100 ml of acetic acid-sodium acetate buffer solution at pH 4 at room temperature;
[0095] S2. Weigh 3 mg of α-glucosidase derived from Bacillus licheniformis and dissolve it in 5 ml of acetic acid-sodium acetate buffer buffer solution with pH 4 to obtain α-glucosidase solution;
[0096] S3. Measure 2 ml of the maltotetraose solution in step S1 into the α-glucosidase solution described in step S2, the volume ratio of the two solutions is 1:1, stir at 1500 rpm, and react in a water bath at 35°C for 35 minutes;
[0097] S4. Place the solution obtained in step S3 in boiling water and boil for 7 minutes, cool to 30°C to inactivate α-glucosidase, and freeze and centrifuge at high speed (4°C, 20,000 rpm, 5 minutes);
[0098] S5. Centrifuge the supernatant obtained in step S4, take 0.3 milliliters, put it into a glass capillary (inner diameter 1.5 millimeters, length 15 centimeters), and seal at both ends;
[0099] S6. the ...
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