Glutatione ridotto Solarbio (GSH) Kit di analisi del contenuto

Glutatione ridotto (GSH) Kit di analisi del contenuto

Nota: Prendi due o tre campioni diversi per la previsione prima del test.

Attrezzatura operativa: Spectrophotometer/Microplate Reader

Numero di catalogo: BC1175

Misurare: 100T/96S

Componenti:

Reagente I: 100 ml×1. Conservare a 4 ℃.

Reagente II: 20 ml×1. Conservare a 4 ℃.

Reagente III: 8 ml×1. Conservare a 4 ℃, protect from light.

Standard: Polvere 10 mg×1. Conservare a 4 ℃, protect from light.

Descrizione del prodotto

Glutathione is a natural tripeptide composed of glutamic acid (Glu), cysteine (Cys) and glycine (Gly). It is a kind of compound containing sulfhydryl group (-SH), which widely exists in animal tissue, plant tissue, microorganism, and yeast. Glutathione can react with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to produce 2-nitro-5-mercaptobenzoic acid and glutathione disulfide (GSSG). 2-nitro-5-mercaptobenzoic acid is a yellow product, with the maximum absorption at 412 nm.

Technical Specifications

Minimum Detection Limit：3.763 μg/mL

Linear Range：12.5-400 μg/mL

Reagenti e attrezzature necessari ma non forniti

Analytical balance, mortaio/omogeneizzatore, low temperature centrifuge, bagnomaria, pipetta regolabile, spectrophotometer/microplate reader, micro glass cuvette or 96 well flat-bottom plate and distilled water.

Procedura

IO. preparazione del campione

1. Campione di tessuto

Wash fresh tissues with PBS for twice, then add 0.1 g of animal/plant tissue into homogenizer (the homogenizer has been rinsed with Reagent I and placed on ice before use). Aggiungere 1 ml di reagente I (the proportion of tissue and Reagents can be kept constant), macinazione completa su ghiaccio (using liquid nitrogen will have a better grinding effect). Centrifugare a 8000 ×g per 10 minuti a 4 ℃, take the supernatant and place it at 4℃ for test. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

2. Blood sample

Plasma: Sample is centrifuged at 600 ×g per 10 minuti a 4 ℃. Absorbing the upper plasma into another tube with adding the same volume Reagent I. Centrifugare a 8000 ×g per 10 minuti a 4 ℃, take the supernatant and place it at 4℃for test. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

Blood cell: Sample is centrifuged at 600 ×g per 10 minuti a 4 ℃. Discarding the upper plasma, wash with three times volume of PBS for 3 volte (re-suspend blood cell with PBS, centrifugare a 600 ×g per 10 minuti), add equal volume of Reagent I. After mixing, it is placed at 4℃ for 10 minuti. Centrifugare a 8000 ×g per 10 minuti, take the supernatant and place it at 4℃ for test. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

3. Cell campione

Harvesting cell should not less than 106,then wash it with PBS for twice (re-suspend cell with PBS, centrifugare a 600 ×g per 10 minuti). The volume of Reagent I added is three times the volume of cell precipitation to re-suspend the cells. Repeated freezing and thawing 2–3 times (It is suggested that frozen in liquid nitrogen, dissolved in 37℃ water bath). Centrifugare a 8000 ×g per 10 minuti, take the supernatant and place it at 4℃ for test. (If the test cannot be completed temporarily, the supernatant can be stored at -80℃ for 10 days.)

II. Procedura

1. Preheat spectrophotometer/microplate reader for 30 minuti, regolare la lunghezza d'onda su 412 nm, azzerare con il distillato
2. Preheat Reagent II in 37℃ (mammal cell) o 25 ℃ (altre specie) bagnomaria per 30
3. Blank tube determination: take micro glass cuvette, aggiungere 20 µl di acqua distillata, 140 μL di Reagente II, 40 μL of Reagent III in turn, mescolare bene, place for 2 minuti, and measure 412 nm absorbance AB.
4. Making standard curve

Weigh 1 mg of standard and dissolve it with 1 mL of distilled water to obtain the concentration of 1 mg/ml. Take the appropriate solution to prepare the standards with the concentration of 200 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL and 12.5 μg/mL (Dilute Reagent I ten times before diluting the standard solution).

Take a 1.5 mL EP tube and add 20 μL of standard, 140 μL of Reagent II and 40 μL of Reagent III in turn. After each tube is evenly mixed, it is allowed to stand for 2 minuti. Misurare l'assorbanza a 412 nm, and absorbance minus AB as abscissa. Make the standard curve according to the absorbance (X) and concentration (y, μg/mL).

5. Sample tube test: take micro glass cuvette, aggiungere 20 μL of sample, 140 μL di Reagente II, 40 μL of Reagent III in turn, mescolare bene, and then stand for 2 minutes to test the absorbance ATat 412 nm, ΔA = AT – AB.
6. The operation of the microplate reader is the same as that of the spectrophotometer, and the operation is as fast as

III. Calcoli

According to the standard curve, take sample ΔA into the formula(X), and calculate the sample concentration y (μg/mL).

• Concentrazione proteica

GSH (μg/mg prot)=y×VRV÷VRV÷Cpr =y÷Cpr

• Peso del campione

GSH (μg/g)=y×VRV÷(VRV÷VSV×W)= y÷W

• Quantità di celle

GSH (μg/104cell)=y×VRV÷(VRV÷VSV×N)= y÷N

• Solution volume GSH (μg/mL)=2y

N: Quantità di celle, 106;

VSV: Volume totale del surnatante, 1 ml；

la corda: Supernatant volume added into the reaction system, 20 μL=0.02 mL； W: Peso del campione, G;

Cpr: Concentrazione proteica nel surnatante, mg/ml.

2: The volume of plasma (blood cells) is diluted by one time.

Nota:

1. The sample needs to be homogenized completely. If the test cannot be completed temporarily, it can be stored at-80℃.
2. Standard: Reduced glutathione is prepared when the solution will be
3. If the GSH content in the sample is uncertain, Dilute the sample for several gradients before
4. Because Reagent I contain protein precipitant, the supernatant cannot be used for protein concentration determination. If the protein content needs to be determined, take another tissue.

Riferimento:

• Alpert A J, Gilbert H F. Detection of oxidized and reduced glutathione with a recycling postcolumn reaction[J]. Analytical biochemistry, 1985, 144(2):553-562.
• Owens C W I, Belcher R V. A colorimetric micro-method for the determination of glutathione[J]. Biochemical Journal, 1965, 94(3):

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