Non-Protein Sulfhydryl Content Assay Kit
enzyme labeling method
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| Performance test | PASS |
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Non-Protein Sulfhydryl Content Assay Kit - Microplate Method
Product Code: 112906
Product Introduction
Thiols in organisms mainly include non-protein thiols and protein thiols. Thiol compounds play important detoxification roles in vivo and are physiologically important for self-regulation.
The thiol group reacts with 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB) to form a yellow compound with a maximum absorption peak at 412 nm.
Reference test result: 2-fold diluted porcine liver sample. OD412 nm: Control 0.064; Sample 0.314/0.323. Actual readings may vary depending on the instrument and test conditions. These data are for reference only.
Package Contents and Storage
Pack size: 100T
| Item Code | Component | Volume / Amount | Storage |
|---|---|---|---|
| 112906.1 | Reagent I | 20 mL | 2-8℃ |
| 112906.2 | Reagent II | 0.75 mL | Protect from light, 2-8℃ |
| 112906.3 | Extraction Solution | 110 mL | Protect from light, 2-8℃ |
| 112906.4 | Standard | 10 mg | Protect from light, 2-8℃ |
| 112906.m | Manual | 1 copy | - |
Quality Standards and Safety Instructions
| Raw Material and Packaging Name | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent I | —— | —— |
| Reagent II | —— | —— |
| Extraction Solution | —— | —— |
| Standard | —— | —— |
Transportation and Storage Conditions
Transportation: This product is shipped with ice packs.
Storage: Store according to the instructions above.
Shelf life: 180 days.
Instructions for Use
1. Sample Preparation
- Tissue samples: Use a tissue mass (g) to extraction solution volume (mL) ratio of 1:5 to 1:10. It is recommended to weigh about 0.1 g tissue and add 1 mL extraction solution. Homogenize in an ice bath, then centrifuge at 8000 g, 4℃ for 10 min. Collect the supernatant and keep it on ice for testing.
- Serum or culture medium: Mix 0.5 mL sample with 0.5 mL extraction solution. Mix well and let stand at room temperature for 10 min, then centrifuge at 8000 g, 4℃ for 10 min. Collect the supernatant and keep it on ice for testing.
2. Reagent Preparation
Standard: The standard contains 10 mg reduced glutathione. Before use, add 1.3 mL extraction solution to dissolve it and prepare a 25 µmol/mL standard solution. Store at 2-8℃ for 4 weeks.
3. Procedure
- Preheat the microplate reader for 30 min, set the wavelength to 412 nm, and zero with distilled water.
- Dilute the 25 µmol/mL standard solution with extraction solution to prepare standard solutions of 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, and 0.0078125 µmol/mL.
Standard Solution Dilution Table
| No. | Concentration Before Dilution (µmol/mL) | Standard Solution Volume (µL) | Extraction Solution Volume (µL) | Concentration After Dilution (µmol/mL) |
|---|---|---|---|---|
| 1 | 25 | 8 | 192 | 1 |
| 2 | 1 | 100 | 100 | 0.5 |
| 3 | 0.5 | 100 | 100 | 0.25 |
| 4 | 0.25 | 100 | 100 | 0.125 |
| 5 | 0.125 | 100 | 100 | 0.0625 |
| 6 | 0.0625 | 100 | 100 | 0.03125 |
| 7 | 0.03125 | 100 | 100 | 0.015625 |
| 8 | 0.015625 | 100 | 100 | 0.0078125 |
Each standard tube in the following experiment requires 40 µL standard solution. Do not directly measure absorbance in this step.
4. Assay Procedure
| Component | Control Tube | Test Tube | Standard Tube | Blank Tube |
|---|---|---|---|---|
| Sample (µL) | 40 | 40 | - | - |
| Standard (µL) | - | - | 40 | - |
| Distilled Water (µL) | - | - | - | 40 |
| Reagent I (µL) | 150 | 150 | 150 | 150 |
| Reagent II (µL) | - | 10 | 10 | - |
| Anhydrous Ethanol (µL) | 10 | - | - | 10 |
Mix well, incubate at 25℃ for 10 min, then measure the absorbance at 412 nm. Record the values as Acontrol, Aassay, Astandard, and Ablank.
Calculate ΔA = Aassay- Acontrol, and ΔAstandard= Astandard- Ablank.
For each assay tube, set one control tube. The standard curve and blank tube only need to be measured 1-2 times.
5. Calculation of Non-Protein Sulfhydryl Content
5.1 Standard curve: Plot the concentration of the standard tube (X, µmol/mL) against absorbance (Y, ΔAstandard) to establish the standard curve. Use the sample ΔA value (Y, ΔA) in the standard curve equation to calculate sample concentration X (µmol/mL).
5.2 Calculation formulas:
- Calculated by sample mass: Non-protein sulfhydryl content (µmol/g mass) = X × Vextract÷ W × F = X ÷ W × F
- Calculated by serum (plasma) or other liquid volume: Non-protein sulfhydryl content (µmol/mL) = X × (Vliquid extract+ Vliquid) ÷ Vliquid× F = 2 × X × F
- Calculated by protein concentration: Non-protein sulfhydryl content (µmol/mg prot) = X × Vextract÷ (Cpr × Vextract) × F = X ÷ Cpr × F
| Symbol | Meaning |
|---|---|
| Vextract | Total volume of sample extract, 1 mL |
| W | Sample mass, g |
| Cpr | Sample protein concentration, mg/mL |
| Vliquid extract | Total volume of liquid sample extract, 0.5 mL |
| Vliquid | Serum (plasma) or other liquid volume, 0.5 mL |
| F | Sample dilution factor |
Precautions
- Before the formal assay, select 2-3 samples expected to show large differences for preliminary testing. This 100T kit can test 48 samples.
- Required instruments and supplies: benchtop centrifuge, microplate reader, constant-temperature water bath, 96-well plate, adjustable pipette, mortar/homogenizer, anhydrous ethanol, ice, and distilled water.
- The linear detection range of this kit is 0.0078125-1 µmol/mL.
- If the measured absorbance exceeds the linear range, increase the sample volume or dilute the sample before measurement.
- The extract contains a protein precipitant, so the supernatant cannot be used for protein concentration determination. If protein content must be measured, use a separate tissue sample.
Appendix
The standard curve is more accurate when prepared by the user. Based on the operation table above, users may use the standard curve formula or the absorbance values of each standard well to plot a standard curve (R2≥ 0.99) and obtain the formula for sample calculation.
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