Reduced glutathione (GSH) kit instructions

Reduced glutathione ( GSH ) kit instructions

Spectrophotometry 50 tubes / 48 samples

Note: Before the formal measurement, select 2-3 samples with large expected differences to make predictions.

Measuring significance:

GSH is the most important anti-oxidation sulfhydryl substance in cells, and plays an important role in antioxidant, protein sulfhydryl protection and amino acid transmembrane transport. The ratio of reduced to oxidized (GSH/GSSG) is the main dynamic indicator of cellular redox status. Therefore, measuring the intracellular GSH and GSSG content and the GSH/GSSG ratio can reflect the redox state of the cells.

Measuring principle:

DTNB reacts with GSH to form a complex with a characteristic absorption peak at 412 nm; its absorbance is proportional to the GSH content.

Bring your own instruments and supplies:

Visible spectrophotometer, cryogenic centrifuge, water bath, adjustable pipette, 1 mL glass cuvette and distilled water.

Reagent composition and formulation:

Reagent 1: Liquid × 1 bottle, stored at 4 ° C.

Reagent 2: Liquid × 1 bottle, stored at 4 ° C.

Reagent 3: Liquid × 1 bottle, stored at 4 ° C protected from light.

Crude enzyme extraction:

1. Tissue: According to the tissue mass (g): the reagent volume (mL) is 1:5~10 (recommended to weigh about 0.1g tissue, add 1mL reagent one) for ice bath homogenization. 8000g, centrifuged at 4 ° C for 10min, take the supernatant and set it on the ice for testing.

2. Bacteria and fungi: According to the number of cells (104): the ratio of reagent volume (mL) is 500~1000:1 (recommended 5 million cells to add 1mL reagent), and the cells are broken by ultrasonic wave in ice bath (power 300w, ultrasound 3) Seconds, 7 seconds apart, total time 3 min); then 8000 g, 4 ° C, centrifuged for 10 min, the supernatant was placed on ice for testing.

3. Liquid such as serum: Direct measurement.

GSH measurement operation:

1. The spectrophotometer is preheated for 30 minutes, the wavelength is adjusted to 412 nm, and the distilled water is zeroed.

2. Reagent II was incubated at 25 ° C (general species) or 37 ° C (mammal) water bath for 30 min.

3. Blank tube: Take 1mL glass cuvette, add 100μL distilled water , 700μL reagent 2, 200μL reagent three, mix and let stand for 2min, then measure 412 nm absorbance A1.

4. Measuring tube: Take 1 mL glass cuvette, add 100 μL of supernatant , 700 μL of reagent 2, and 200 μL of reagent three in sequence, and mix and let stand for 2 min to measure absorbance A2 at 412 nm.

Note: The blank tube only needs to be measured once.

GSH content calculation formula:

GSH standard curve formula: y=1.5 x (x is GSH concentration, μ mol /mL; y is absorbance)

GSH calculation:

(1) Calculated by protein concentration

GSH(μ mol/mg prot)=(A2-A1) ÷1.5×V ÷(V sample×Cpr )=0.667×(A2-A1) ÷Cpr

(2) Calculated by sample fresh weight

GSH (μ mol / g fresh weight) = (A2-A1) ÷ 1.5 × V sample V (V sample ÷ V sample total × W) = 0.667 × (A2-A1) ÷ W

(3) Calculated by cell number

GSH(μ mol /104 cell)=(A2-A1) ÷1.5×V ÷(V-like ÷V-like total × cell number)=0.667×(A2-A1) ÷ cell number (4) Calculated by liquid volume

GSH (μ mol / mL) = (A2-A1) ÷ 1.5 × V sample ÷ V sample = 0.667 × (A2-A1)

V sample total: total volume of supernatant, 1 mL; V sample: volume of supernatant added to the reaction system, 100 μL = 0.1 mL; W: sample mass, g; Cpr: supernatant protein concentration, mg / mL;

Precautions:

1. Reagent 1 contains a protein precipitant, so the supernatant cannot be used for protein concentration determination.

2. The minimum detection limit is 0.01mmol/L.