Alpha-Amylase Inhibitor Screening Kit (Colorimetric)

• Cat.No.: KITZ-005
• Product Overview: Alpha-Amylase Inhibitor Screening Kit (Colorimetric) can be used to screen/characterize potential inhibitors of Alpha-Amylase.

Specification

• Description: Alpha-Amylase enzyme is responsible for the breakdown of complex carbohydrates in humans. Thus, inhibition of α-amylase could be considered as a strategy for the treatment of disorders related to carbohydrate uptake, such as obesity, diabetes, dental cavities and periodontal diseases. In this kit human Alpha-Amylase hydrolyzes the synthetic substrate, yielding smaller fragments containing the chromophore (pNP; OD = 405 nm). A potent, specific inhibitor is also included in the kit. The one-step assay is simple and can be completed within 1 hour.
• Storage: On receipt entire assay kit should be stored at -20°C. Kit has a storage time of 1 year from receipt, providing components have not been reconstituted.
• Kit Components: Alpha-Amylase Assay Buffer 1 x 55ml
  Alpha-Amylase Inhibitor 1 x 100µl
  Alpha-Amylase Substrate 1 vial
  Alpha-Amylase, Human Saliva 1 vial
• Materials Required but Not Supplied: 96-well clear plate with flat bottom
  Multi-well spectrophotometer (ELISA reader)
• Detection method: Colorimetric, OD = 405 nm
• Assay Protocol: Reagent Preparation:
  Before using the kit, spin the tubes prior to opening.
  1.Alpha-Amylase Assay Buffer: Warm to room temperature (RT) before use. Store at 4°C or -20°C.
  2.Alpha-Amylase Substrate: Reconstitute with 100 µL Alpha-Amylase Assay Buffer to prepare stock solution. Aliquot Stock Solution in 10 µL aliquots and store at -20°C. Use aliquot once only.
  3.Alpha-Amylase, Human Saliva: Reconstitute with 100 µL Alpha-Amylase Assay Buffer to prepare stock solution. Aliquot Stock Solution in 10 µL aliquots and store at -20°C. Use aliquot once only.
  4.Alpha-Amylase Inhibitor: Ready to use. Keep on ice while in use.
  Assay Protocol:
  Screening Compounds, Inhibitor Control & Background Control preparations:
  1.Dissolve test samples 100X in a proper solvent.
  2.Further dilute to 3X with Alpha-Amylase Assay Buffer. Mix well.
  -Test Samples [S]: Add 50 µL of Diluted test samples (3X) to designated wells of a clear 96 well microplate.
  -Inhibitor Control [IC]: Add 10 µL of reconstituted Alpha-Amylase inhibitor to 40 µL Assay Buffer in designated wells.
  -Enzyme Control [EC]: Add 50 µL of Alpha-Amylase Assay Buffer to designated wells.
  -Background Control [BC]: Add 100 µL of Alpha-Amylase Assay Buffer to designated wells.
  -Solvent Control [SC]: Add 50 µL of 3X Solvent (the same concentration of solvent as in the diluted Test Samples) in Alpha-Amylase Assay Buffer to designated wells.
  -IC50 estimation (Optional): prepare several dilutions of candidate(s) in Alpha-Amylase Assay Buffer maintaining Solvent Concentration constant for all concentrations. Add 50 µL of each dilution into designated individual wells.
  Δ Note: Various organic solvents may reduce the Alpha-Amylase enzymatic activity. Prepare parallel well(s) as Solvent Control [SC] to test the effect of the solvent on Alpha-Amylase-Amylase enzymatic activity. If [SC] slope is significantly different when compared to [EC], use [SC] values to determine effect of the respective tested compounds (see Step 5-Calculations).
  Alpha-Amylase Enzyme Solution Preparation:
  1. Dilute Alpha-Amylase stock by adding 490 μL of Alpha-Amylase Assay Buffer into 10 μL of Alpha-Amylase Enzyme.
  2. Mix thoroughly by pipetting up and down.
  3. Add 50 μL of diluted Alpha-Amylase Solution to each well containing Test Sample(s) [S], Inhibitor Control [IC], Enzyme Control [EC] and Solvent Control [SC].
  4. Avoid introducing any bubbles into the wells. Mix well.
  5. Incubate at room temperature (RT) for 10 minutes, protected from light.
  Δ Note: Do not store Diluted Alpha-Amylase Enzyme Solution. Discard unused solution.
  Alpha-Amylase Substrate Mix Preparation:
  1. Dilute Substrate by adding 490 µL of Alpha-Amylase Assay Buffer into 10 µL of Alpha Amylase Substrate.
  2. Mix thoroughly by pipetting up and down.
  3. Add 50 µL of diluted Alpha-Amylase Substrate to each well containing Test Sample(s) [S], Inhibitor Control [IC], Enzyme Control [EC], Background Control [BC] and Solvent Control [SC].
  4. Avoid introducing any bubbles into the wells. Mix well.
  Measurement:
  Measure absorbance at OD=405 nm in kinetic mode for 20-25 min at room temperature. Choose two time points (t1 and t2) in the linear range of the plot and obtain the corresponding values for the absorbance (OD1 and OD2).
  Calculation:
  1. Subtract the reading of Background Control [BC] from all test samples [S], Enzyme Control [EC], and Solvent Control [SC].
  2. Calculate the slope of all wells by dividing the net ΔOD (OD2 - OD1) over time Δt (t2 - t1).
  3. If [SC] slope is significantly different when compared to [EC], use [SC] values to determine the inhibitory effect of tested compound(s).