Reduced Glutathione Assay Kit (Spectrophotometry)

Taking 50T/48S packing for example:

Note: Before formal testing, it is essential to take 2-3 samples with large expected differences for preliminary testing.

1. Preparation of Lab Instruments

Visible spectrophotometer, 1 mL glass cuvette, distilled water, low-temperature centrifuge, homogenizer/mortar, water bath, and adjustable pipettes.

2. Sample Extraction

Blood sample:

Plasma: Centrifuge the collected anticoagulated blood at 600g for 10 minutes at 4 °C. Transfer the upper plasma layer to a new tube, add an equal volume of CB0130S-A, and centrifuge at 8000g for 10 minutes at 4°C. Transfer the supernatant to a new tube and store at 4°C for testing; if testing cannot be completed immediately, store at -80°C (stable for up to 10 days).

Blood Cells: Centrifuge the collected anticoagulated blood at 600g for 10 minutes at 4°C. Discard the upper plasma layer and wash 3 times with 3 volumes of PBS (resuspend blood cells in PBS, centrifuge at 600g for 10 minutes). Add an equal volume of CB0130S-A, mix well, and incubate at 4°C for 10 minutes. Centrifuge at 8000g for 10 minutes, collect the supernatant, and store at 4°C for testing; if testing cannot be completed immediately, store at -80°C (stable for up to 10 days).

Cell or Tissue sample:

Cells: Collect no less than 10^6 cells. Wash the cells twice with PBS (resuspend cells in PBS, centrifuge at 600g for 10 minutes). Add 3 times the cell pellet volume of CB0130S-A to resuspend the cells, and subject to 2-3 freeze-thaw cycles (freeze in liquid nitrogen, thaw in a 37°C water bath). Centrifuge at 8000g for 10 minutes, collect the supernatant, and store at 4°C for testing; if testing cannot be completed immediately, store at -80°C (stable for up to 10 days).

Tissue: Rinse fresh tissue twice with PBS, then weigh 0.1 g of animal or plant tissue. Place into a homogenizer pre-rinsed with CB0130S-A (pre-cool the homogenizer on ice). Add 1 mL of CB0130S-A (maintain the tissue/CB0130S-A ratio) and rapidly grind thoroughly on ice (grinding with liquid nitrogen yields better results). Centrifuge at 8000g for 10 minutes at 4°C. Collect the supernatant and store at 4°C for testing; if testing cannot be completed immediately, store at -80°C (stable for up to 10 days).

3. Assay Procedure

Preheat the spectrophotometer or microplate reader for 30 minutes, adjust the wavelength to 412 nm, and zero with distilled water.

Incubate CB0130S-B in a water bath at 25°C (for general species) or 37°C (for mammals) for 30 minutes.

Standard Curve Preparation:

Weigh 1 mg of standard and dissolve in 1 mL of distilled water to prepare a 1 mg/mL stock solution (prepare fresh before use).

Dilute the standard with CB0130S-A to concentrations of 100 µg/mL, 75 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, and 0 µg/mL.

Sequentially add 100 µL of each standard concentration, 700 µL of CB0130S-B, and 200 µL of CB0130S-C to EP tubes. Mix well and let stand for 2 minutes. Measure the absorbance at 412 nm and plot the standard curve based on absorbance (x) and concentration (y, µg/mL).

Add the following reagents to EP tubes:

4. Calculation

Based on the standard curve, substitute the sample ΔA into the formula as (x) to calculate the sample concentration y (µg/mL).

Calculated by Protein Concentration

GSH(μg/mg prot)=y×Vs÷(Vs×Cpr)=y÷Cpr

Calculated by Sample Mass

GSH(μg/g mass)=y×Vs ÷(Vs ÷Vt ×W)=y÷W

Calculated by Cell Number

GSH(μg/10^6 cells)=y×Vs ÷(Vs ÷Vt ×CN)=y÷CN

Calculated by Plasma (Blood Cell) Volume

GSH(μg/mL)=2y

Note:

Vt: Total volume of supernatant, 1 mL.

Vs: Volume of supernatant added to the reaction system, 100 µL = 0.1 mL.

W: Sample mass, g.

Cpr: Supernatant protein concentration, mg/mL.

CN: Cell number, Measured in units of 10^6

2: The plasma (blood cell) volume is diluted twofold.

1.CB0130S-A contains a protein precipitant; therefore, the supernatant cannot be used for protein concentration assays. If protein content needs to be measured, a separate tissue sample must be prepared.

2.Sample processing requires complete homogenization. If the measurement cannot be completed on the same day, store at -80°C for no more than 10 days.

3.If the measured absorbance exceeds the linear range, increase the sample volume or dilute the sample before testing.

4.The product is for R&D use only, not for diagnostic procedures, food, drug, household or other uses.

5.Please wear a lab coat and disposable gloves.

• CB0130S-Instruction Manual(341 KB)