Total Thiol Content Assay Kit (DTNB, Colorimetric Method)

Cat. No.: T1515982

GRADE & PURITY BioReagent

Descripción general

Sulfhydryl groups in organisms mainly include glutathione sulfhydryl (GSH) and protein sulfhydryl groups. The former not only repairs oxidatively damaged proteins but also participates in the scavenging of reactive oxygen species, while the latter plays an important role in maintaining protein conformation. By measuring total sulfhydryl content and GSH content, protein sulfhydryl content can be indirectly determined.

Detection Principle:

Sulfhydryl groups react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to form a yellow compound with a maximum absorption peak at 412 nm.

Note: For research use only. Not for clinical diagnosis or other purposes.

Standard (T1515982D) Precautions:

• Use this standard if you need to prepare a new standard curve.

• Prepare according to the standard curve preparation instructions.

• Dissolved standard should be used within one week.

Reagents, consumables and Equipments not provided

• Spectrophotometer (capable of measuring absorbance at 412 nm)
• Mortar and pestle (or homogenizer), ice box (ice maker), benchtop centrifuge, adjustable pipettes, water bath (oven, incubator, heating block)
• 1 mL cuvettes, centrifuge tubes
• Distilled water (deionized water or ultrapure water acceptable)

Protocol (for reference only):

1. Sample Preparation

1.1 Tissue samples:

Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 10,000 rpm for 10 min at 4°C. Collect the supernatant and keep on ice until use.

Notes:

① If available, grind tissue under liquid nitrogen before adding Extraction Buffer and homogenizing on ice.

② According to research needs, extraction can be performed at a ratio of tissue mass (g) to Extraction Buffer volume (mL) = 1:10.

1.2 Liquid samples:

Clear liquid samples can be directly measured. If turbid, centrifuge and use the supernatant for measurement.

2. Assay Procedure

2.1 Preheat the visible spectrophotometer for 30 min (or allow the instrument to complete its self-check), set the wavelength to 412 nm, and zero with distilled water.

2.2 All reagents should be warmed to room temperature or in a 25°C water bath for 10 min before use.

2.3 Add the following reagents to an EP tube or a 1 mL glass cuvette (1 cm light path) in order:

Reagent (μL)	Assay Tube	Control Tube
Sample	120	120
Reagent 1	480	560
Reagent 2	80	—

Mix well, let stand at 25°C for 2 min, then measure the absorbance at 412 nm. ΔA = A_Assay – A_Control (a self-control for each sample is required).

Notes:

① If a white turbidity appears after adding Reagent 2, mix the sample immediately to restore clarity.

② If A_Assay > 1.5, reduce the sample volume V₁ (e.g., from 30 μL to 10 μL) and increase Reagent 1 accordingly, or dilute the supernatant with distilled water before measurement. The changed V₁ and dilution factor (D) must be included in the calculation.

3. Calculation

3.1 Standard curve equation: y = 3.4274x + 0.0008, where x = standard concentration (μmol/mL), y = ΔA.

3.2 Calculation of total sulfhydryl content in samples:

(1) Based on sample mass (fresh weight):

Total sulfhydryl content (μmol/g fresh weight) = [(ΔA – 0.0008) ÷ 3.4274 × V₁] ÷ (W × V₁ ÷ V) × D

= 0.2918 × (ΔA – 0.0008) ÷ W × D

(2) Based on protein concentration:

Total sulfhydryl content (μmol/mg prot) = [(ΔA – 0.0008) ÷ 3.4274 × V₁] ÷ (C_pr × V₁ ÷ V) × D

= 0.2918 × (ΔA – 0.0008) ÷ C_pr × D

(3) Based on liquid sample volume:

Total sulfhydryl content (μmol/mL) = [(ΔA – 0.0008) ÷ 3.4274 × V₁] ÷ V₁ × D

= 0.2918 × (ΔA – 0.0008) × D

(4) Based on bacterial/cell count:

Total sulfhydryl content (μmol/10⁴ cells) = [(ΔA – 0.0008) ÷ 3.4274 × V₁] ÷ (500 × V₁ ÷ V) × D

= 0.2918 × (ΔA – 0.0008) ÷ 500 × D

Parameters:

V = volume of Extraction Buffer added = 1 mL

V₁ = sample volume added = 0.12 mL

W = sample mass (g)

Molecular weight of GSH = 307.3

D = dilution factor (if undiluted, D = 1)

500 = total number of bacteria or cells (×10⁴)

C_pr = protein concentration in the supernatant (mg/mL). It is recommended to use Aladdin B665595 BCA Protein Assay Kit or R1491648 Ready-to-Use BCA Protein Assay Kit.

Standard Curve Preparation

1. Preparing a standard curve is not mandatory. Users may either generate a standard curve or directly use the formula provided in the manual for calculation.

2. Prepare standard stock solution (1 μmol/mL): Add 2 mL of distilled water to the standard vial and dissolve completely. This yields a 1 μmol/mL standard stock solution.

3. Dilute the stock solution with distilled water to six concentration gradients, e.g., 0, 0.1, 0.2, 0.3, 0.4, 0.5 μmol/mL. Concentrations may be adjusted according to actual samples.

4. Standard dilution reference table:

Pipette 500 μL of standard stock solution, add 500 μL distilled water, mix to obtain 0.5 μmol/mL diluted standard.

Standard concentration (μmol/mL)	0	0.1	0.2	0.3	0.4	0.5
Diluted standard (μL)	0	40	80	120	160	200
Water (μL)	200	160	120	80	40	0

Mix each standard tube well before use.

5. Operate according to the Assay Tube addition table, and generate the standard curve passing through zero.

Reagent (μL)	Standard Tube	Zero Tube (once only)
Standard	120	—
Distilled water	—	120
Reagent 1	480	480
Reagent 2	80	80

Mix, let stand at 25°C for 2 min, transfer the entire volume to a 1 mL cuvette, and measure absorbance at 412 nm. ΔA = A_Standard – A_Zero.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light

Enviado en

Wet ice

Estabilidad y almacenamiento

Store at 2-8℃ long term (6 months). Store in the dark.

Contents & Storage

T1515982	Component	100T/48S	Storage
T1515982A	Extraction Buffer	60 mL	2-8℃.
T1515982B	Reagent 1	55 mL	2-8℃.
T1515982C	Reagent 2	4 mL	2-8℃. Store in the dark.
T1515982D	Standard	1 EA	2-8℃.