UV-VIS Anti-Trypsic (Anti-Trypsin) Activity Measurement
- Product Code: 125877
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
-
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.
-
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.
-
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.
- In each cuvette, add the following components:
-
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.
-
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.
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
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
-
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.
-
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.
-
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.
- In each cuvette, add the following components:
-
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.
-
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.
Mechanism | - |
Appearance | - |
Longevity | - |
Strength | - |
Storage | - |
Shelf Life | - |
Allergen(s) | - |
Dosage (Range) | - |
Recommended Dosage | - |
Dosage (Per Day) | - |
Recommended Dosage (Per Day) | - |
Mix Method | - |
Heat Resistance | - |
Stable in pH range | - |
Solubility | - |
Product Types | - |
INCI | - |
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. |
Cart
No products