UV-VIS Anti-Trypsic (Anti-Trypsin) Activity Measurement

  • Product Code: 125877

UV-VIS Anti-Trypsic (Anti-Trypsin) Activity Measurement

฿2,990.00

Objective

To measure the anti-trypsin activity of a plant extract or compound using UV-VIS spectroscopy, assessing its ability to inhibit the activity of trypsin on a substrate (such as BAPNA or casein).

Materials

  • Trypsin enzyme: Bovine trypsin, typically from pancreas (lyophilized powder)
  • Substrate: N-Benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) or casein
  • Buffer: Tris-HCl (50 mM, pH 7.8) or Phosphate-buffered saline (PBS) (50 mM, pH 7.4)
  • Plant extract/compound: Test sample with potential anti-trypsin activity
  • Dimethyl sulfoxide (DMSO) or another suitable solvent (if required to dissolve plant extract)
  • UV-VIS spectrophotometer
  • Cuvettes: 1 cm path length
  • Water bath/incubator: Set to 37°C
  • Stop solution: Acetic acid or trichloroacetic acid (TCA, 10%)

Method

  1. Preparation of Solutions

    • Trypsin Stock Solution: Dissolve trypsin in Tris-HCl buffer to a final concentration of 1 mg/mL. Prepare aliquots and store at −20°C.
    • BAPNA Substrate Solution: Dissolve BAPNA (1 mM) in Tris-HCl buffer. Alternatively, dissolve casein (1%) in PBS for casein assay.
    • Plant Extract Solution: Dissolve the plant extract in an appropriate solvent (such as DMSO) to a final concentration of 1 mg/mL. Prepare a range of dilutions for IC50 determination.
  2. Control and Test Samples

    • Blank Control: Contains buffer and substrate but no trypsin or plant extract.
    • Positive Control: Contains trypsin and substrate without plant extract to measure maximum enzyme activity.
    • Test Sample: Contains trypsin, substrate, and various concentrations of plant extract to determine its inhibitory effect.
  3. Incubation

    • In each cuvette, add the following components:
      • Blank Control: 900 µL buffer + 100 µL substrate
      • Positive Control: 800 µL buffer + 100 µL substrate + 100 µL trypsin
      • Test Sample: 700 µL buffer + 100 µL substrate + 100 µL trypsin + 100 µL plant extract solution (vary concentrations of plant extract for inhibition studies)
    • Mix gently and incubate at 37°C for 30 minutes.
  4. Reaction Termination

    • After the incubation period, add 100 µL of stop solution (10% TCA) to terminate the reaction in each cuvette. This will precipitate the protein in casein assays or stop the release of p-nitroaniline from BAPNA.
  5. UV-VIS Measurement

    • For BAPNA assay, measure the absorbance at 410 nm (corresponding to the yellow color of p-nitroaniline) using a UV-VIS spectrophotometer.
    • For casein assay, measure the absorbance at 280 nm to detect the amount of released tyrosine or other aromatic amino acids from casein digestion.
    • Record absorbance values for each sample.

Notes

  • Ensure that the solvent used for the plant extract does not interfere with enzyme activity or UV-VIS measurements.
  • Maintain all samples at 37°C during incubation for optimal enzyme activity.
  • Replicate each sample in triplicate to ensure reliable results.

Results and Interpretation

  • A decrease in absorbance at 410 nm (for BAPNA) or 280 nm (for casein) in the presence of plant extract indicates trypsin inhibition.
  • The plant extract's ability to inhibit trypsin can be quantified by comparing it to the positive control.

Service Steps
Step Procedure Expected Result
1

Prepare the...

A 1 mg/mL plant extract solution ready for testing.

2

Prepare...

A clear Tris-HCl buffer at 20 mM and pH 7.5.

3

Dissolve...

A trypsin solution at the required concentration (60 µg in the chosen volume) in Tris-HCl buffer.

4

Prepare...

A 1 mM BApNA substrate solution in Tris-HCl buffer with 1% DMSO.

5

Prepare...

A 1 mg/mL AEBSF solution as a known inhibitor of trypsin.And negative control.

6

Pipette 40...

Each well contains 40 µL of trypsin.
7

Pipette 40...

Each well now contains 80 µL total volume: 40 µL trypsin + 40 µL sample/control.

8

Incubate the...

Pre-incubation for trypsin inhibition reaction to occur.

9

Add 100...

Substrate is introduced, final well volume is 180 µL.

10

Incubate the...

Enzymatic hydrolysis of BApNA occurs (or is inhibited), generating color.

11

Add 30%...

Reaction is halted by acidification; color development is stabilized.

12

Measure and...

Absorbance values (OD) for control and sample wells are obtained.

13

Calculate percentage...

Percentage inhibition result for each sample.

You will receive a report for the % Inhibition of Trypsin fields when we provide this service

UV-VIS Anti-Trypsic (Anti-Trypsin) Activity Measurement

UV-VIS Anti-Trypsic (Anti-Trypsin) Activity Measurement

Objective

To measure the anti-trypsin activity of a plant extract or compound using UV-VIS spectroscopy, assessing its ability to inhibit the activity of trypsin on a substrate (such as BAPNA or casein).

Materials

  • Trypsin enzyme: Bovine trypsin, typically from pancreas (lyophilized powder)
  • Substrate: N-Benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) or casein
  • Buffer: Tris-HCl (50 mM, pH 7.8) or Phosphate-buffered saline (PBS) (50 mM, pH 7.4)
  • Plant extract/compound: Test sample with potential anti-trypsin activity
  • Dimethyl sulfoxide (DMSO) or another suitable solvent (if required to dissolve plant extract)
  • UV-VIS spectrophotometer
  • Cuvettes: 1 cm path length
  • Water bath/incubator: Set to 37°C
  • Stop solution: Acetic acid or trichloroacetic acid (TCA, 10%)

Method

  1. Preparation of Solutions

    • Trypsin Stock Solution: Dissolve trypsin in Tris-HCl buffer to a final concentration of 1 mg/mL. Prepare aliquots and store at −20°C.
    • BAPNA Substrate Solution: Dissolve BAPNA (1 mM) in Tris-HCl buffer. Alternatively, dissolve casein (1%) in PBS for casein assay.
    • Plant Extract Solution: Dissolve the plant extract in an appropriate solvent (such as DMSO) to a final concentration of 1 mg/mL. Prepare a range of dilutions for IC50 determination.
  2. Control and Test Samples

    • Blank Control: Contains buffer and substrate but no trypsin or plant extract.
    • Positive Control: Contains trypsin and substrate without plant extract to measure maximum enzyme activity.
    • Test Sample: Contains trypsin, substrate, and various concentrations of plant extract to determine its inhibitory effect.
  3. Incubation

    • In each cuvette, add the following components:
      • Blank Control: 900 µL buffer + 100 µL substrate
      • Positive Control: 800 µL buffer + 100 µL substrate + 100 µL trypsin
      • Test Sample: 700 µL buffer + 100 µL substrate + 100 µL trypsin + 100 µL plant extract solution (vary concentrations of plant extract for inhibition studies)
    • Mix gently and incubate at 37°C for 30 minutes.
  4. Reaction Termination

    • After the incubation period, add 100 µL of stop solution (10% TCA) to terminate the reaction in each cuvette. This will precipitate the protein in casein assays or stop the release of p-nitroaniline from BAPNA.
  5. UV-VIS Measurement

    • For BAPNA assay, measure the absorbance at 410 nm (corresponding to the yellow color of p-nitroaniline) using a UV-VIS spectrophotometer.
    • For casein assay, measure the absorbance at 280 nm to detect the amount of released tyrosine or other aromatic amino acids from casein digestion.
    • Record absorbance values for each sample.

Notes

  • Ensure that the solvent used for the plant extract does not interfere with enzyme activity or UV-VIS measurements.
  • Maintain all samples at 37°C during incubation for optimal enzyme activity.
  • Replicate each sample in triplicate to ensure reliable results.

Results and Interpretation

  • A decrease in absorbance at 410 nm (for BAPNA) or 280 nm (for casein) in the presence of plant extract indicates trypsin inhibition.
  • The plant extract's ability to inhibit trypsin can be quantified by comparing it to the positive control.

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Service Steps
Step Procedure Expected Result
1

Prepare the...

A 1 mg/mL plant extract solution ready for testing.

2

Prepare...

A clear Tris-HCl buffer at 20 mM and pH 7.5.

3

Dissolve...

A trypsin solution at the required concentration (60 µg in the chosen volume) in Tris-HCl buffer.

4

Prepare...

A 1 mM BApNA substrate solution in Tris-HCl buffer with 1% DMSO.

5

Prepare...

A 1 mg/mL AEBSF solution as a known inhibitor of trypsin.And negative control.

6

Pipette 40...

Each well contains 40 µL of trypsin.
7

Pipette 40...

Each well now contains 80 µL total volume: 40 µL trypsin + 40 µL sample/control.

8

Incubate the...

Pre-incubation for trypsin inhibition reaction to occur.

9

Add 100...

Substrate is introduced, final well volume is 180 µL.

10

Incubate the...

Enzymatic hydrolysis of BApNA occurs (or is inhibited), generating color.

11

Add 30%...

Reaction is halted by acidification; color development is stabilized.

12

Measure and...

Absorbance values (OD) for control and sample wells are obtained.

13

Calculate percentage...

Percentage inhibition result for each sample.

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