Pepsin Activity Assay Kit
SKU: E-BC-K844-M-96
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Pepsin Activity Assay Kit
| SKU # | E-BC-K844-M |
| Detection Instrument | Microplate reader (650-670 nm, optimum wavelength: 660 nm) |
| Detection method: | Colorimetric method |
Product Details
Properties
| Sample type | Gastric, intestinal tissue |
| Sensitivity | 0.63 U/mL |
| Detection range | 0.63-94.98 U/mL |
| Detection method |
Colorimetric method |
| Assay type | Enzyme Activity |
| Precision | Average inter-assay CV: 4.8-5.6% | Average intra-assay CV: 1.3-2.2% |
| Storage | -20℃ |
| Valid period | 12 months |
Dilution of Sample
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
| 20% Rat gastric tissue homogenate | 1 |
| 20% Mouse gastric tissue homogenate | 1 |
| 20% Rat intestinal tissue homogenate | 1 |
| 20% Mouse intestinal tissue homogenate | 1 |
Note: The diluent is buffer solution. For the dilution of other sample types, please do pretest to confirm the dilution factor.
Detection Principle
Pepsin can catalyze substrate hydrolysis, and the hydrolysate reacts with the chromogenic agent under alkaline conditions to form a blue substance. The blue substance has a maximum absorption peak at 660 nm, and the color depth is proportional to the activity of pepsin. The enzyme activity can be calculated by OD value
Kit Components & Storage
| Item | Component | Size (96 T) | Storage |
| Reagent 1 | Buffer Solution | 55 mL × 2 vials | -20℃, 12 months |
| Reagent 2 | Precipitating Agent | 50 mL × 1 vial | -20℃, 12 months |
| Reagent 3 | Chromogenic Agent A | 20 mL × 1 vial | -20℃, 12 months |
| Reagent 4 | Chromogenic Agent B | 10 mL × 1 vial | -20℃, 12 months, shading light |
| Reagent 5 | Substrate | Power × 2 vials | -20℃, 12 months, shading light |
| Reagent 6 | Substrate Solvent | 12 mL × 1 vial | -20℃, 12 months |
| Reagent 7 | 1 mg/mL Standard Solution | 1.5 mL × 1 vial | -20℃, 12 months, shading light |
| Microplate | 96 wells | No requirement | |
| Plate Sealer | 2 pieces |
Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.
Technical Data:
Parameter:
Intra-assay Precision
Three rat gastric tissue samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (U/mL) | 3.80 | 46.80 | 73.50 |
| %CV | 2.2 | 1.6 | 1.3 |
Inter-assay Precision
Three rat gastric tissue samples were assayed 20 times in duplicate by three operators to determine precision between assays.
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (U/mL) | 3.80 | 46.80 | 73.50 |
| %CV | 4.8 | 5.6 | 5.2 |
Recovery
Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 95%.
| Standard 1 | Standard 2 | Standard 3 | |
| Expected Conc. (μg/mL) | 15 | 32 | 45.5 |
| Observed Conc. (μg/mL) | 14.7 | 29.8 | 42.8 |
| Recovery rate(%) | 98 | 93 | 94 |
Sensitivity
The analytical sensitivity of the assay is 0.63 U/mL. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.
Standard Curve
As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only:
| Concentration (μg/mL) | 0 | 12 | 18 | 24 | 36 | 42 | 48 | 60 |
| OD Value | 0.041 | 0.163 | 0.222 | 0.285 | 0.411 | 0.476 | 0.53 | 0.647 |
| 0.041 | 0.164 | 0.226 | 0.286 | 0.408 | 0.469 | 0.529 | 0.648 | |
| Average OD | 0.041 | 0.164 | 0.224 | 0.286 | 0.410 | 0.473 | 0.530 | 0.648 |
| Absoluted OD | 0 | 0.123 | 0.183 | 0.245 | 0.369 | 0.432 | 0.489 | 0.647 |