UV-VIS Anti-Trypsic Assay
- Product Code: 125265
UV-VIS Anti-Trypsic Assay
UV-VIS Anti-Trypsic Assay
Materials and Reagents
- Plant extract with antitryptic activity
- Trypsin enzyme solution
- Substrate (e.g., Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPNA))
- Tris-HCl buffer (50 mM, pH 8.0)
- Dimethyl sulfoxide (DMSO) or another suitable solvent for dissolving BAPNA
- UV-VIS spectrophotometer
- Quartz cuvettes
- Distilled water
Preparation of Solutions
- Trypsin Solution: Dissolve trypsin in Tris-HCl buffer to a concentration of 0.01 mg/mL.
- Substrate Solution (BAPNA): Dissolve BAPNA in DMSO to make a 5 mg/mL stock solution. Dilute this stock solution with Tris-HCl buffer to a final concentration of 0.1 mM.
- Plant Extract Solution: Dissolve the plant extract in Tris-HCl buffer. Prepare different concentrations for testing (e.g., 0.1 mg/mL, 0.2 mg/mL, etc.).
Assay Procedure
- Blank Preparation: Mix 2.9 mL of Tris-HCl buffer with 0.1 mL of the substrate solution. This serves as a blank to zero the spectrophotometer.
- Control Reaction: Mix 2.8 mL of Tris-HCl buffer, 0.1 mL of substrate solution, and 0.1 mL of trypsin solution. This will measure the activity of trypsin without inhibition.
- Test Reaction: Mix 2.7 mL of Tris-HCl buffer, 0.1 mL of substrate solution, 0.1 mL of trypsin solution, and 0.1 mL of plant extract solution. Prepare separate test reactions for each concentration of plant extract.
Measurement
- Incubation: Incubate all reaction mixtures at 37°C for 10 minutes.
- Measurement: Measure the absorbance at 410 nm using the UV-VIS spectrophotometer. The increase in absorbance is due to the release of p-nitroaniline from the substrate by trypsin action.
Calculation of Antitryptic Activity
-
Determine the Change in Absorbance:
- ΔAcontrolΔAcontrol = Absorbance of control reaction - Absorbance of blank
- ΔAtestΔAtest = Absorbance of test reaction - Absorbance of blank
-
Inhibition Percentage:
Inhibition (%)=(ΔAcontrol−ΔAtestΔAcontrol)×100Inhibition (%)=(ΔAcontrolΔAcontrol−ΔAtest)×100
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UV-VIS Anti-Trypsic Assay
UV-VIS Anti-Trypsic Assay
UV-VIS Anti-Trypsic Assay
Materials and Reagents
- Plant extract with antitryptic activity
- Trypsin enzyme solution
- Substrate (e.g., Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPNA))
- Tris-HCl buffer (50 mM, pH 8.0)
- Dimethyl sulfoxide (DMSO) or another suitable solvent for dissolving BAPNA
- UV-VIS spectrophotometer
- Quartz cuvettes
- Distilled water
Preparation of Solutions
- Trypsin Solution: Dissolve trypsin in Tris-HCl buffer to a concentration of 0.01 mg/mL.
- Substrate Solution (BAPNA): Dissolve BAPNA in DMSO to make a 5 mg/mL stock solution. Dilute this stock solution with Tris-HCl buffer to a final concentration of 0.1 mM.
- Plant Extract Solution: Dissolve the plant extract in Tris-HCl buffer. Prepare different concentrations for testing (e.g., 0.1 mg/mL, 0.2 mg/mL, etc.).
Assay Procedure
- Blank Preparation: Mix 2.9 mL of Tris-HCl buffer with 0.1 mL of the substrate solution. This serves as a blank to zero the spectrophotometer.
- Control Reaction: Mix 2.8 mL of Tris-HCl buffer, 0.1 mL of substrate solution, and 0.1 mL of trypsin solution. This will measure the activity of trypsin without inhibition.
- Test Reaction: Mix 2.7 mL of Tris-HCl buffer, 0.1 mL of substrate solution, 0.1 mL of trypsin solution, and 0.1 mL of plant extract solution. Prepare separate test reactions for each concentration of plant extract.
Measurement
- Incubation: Incubate all reaction mixtures at 37°C for 10 minutes.
- Measurement: Measure the absorbance at 410 nm using the UV-VIS spectrophotometer. The increase in absorbance is due to the release of p-nitroaniline from the substrate by trypsin action.
Calculation of Antitryptic Activity
-
Determine the Change in Absorbance:
- ΔAcontrolΔAcontrol = Absorbance of control reaction - Absorbance of blank
- ΔAtestΔAtest = Absorbance of test reaction - Absorbance of blank
-
Inhibition Percentage:
Inhibition (%)=(ΔAcontrol−ΔAtestΔAcontrol)×100Inhibition (%)=(ΔAcontrolΔAcontrol−ΔAtest)×100
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