Microscale Reduced Glutathione assay kit

Instruction manual

First Edition (Revised on April, 2016)

[ INTENDED USE ]

The kit is a microscale enzyme labelled method for the in vitro quantitative measurement of reduced glutathione (GSH) activity in cultured cells, tissues, serum, blood plasma, Whole blood etc..

[ REAGENTS AND MATERIALS PROVIDED ]

Reagents	Quantity(96T)	Reagents		Quantity(96T)
Reagent 1	1×20ml	Reagent 2		1×20ml
Reagent 3	1×5ml	Reagent 4	GSH standard powder	3
			Standard solvent stock solution	1×10ml
Instruction manual	1	96T plate		1

[ MATERIALS REQUIRED BUT NOT SUPPLIED ]

1. A microplate reader capable of measuring absorbance at 405nm.

2. Desk centrifuge.

3. Transferpettor and tips.

4. Vortex mixer.

5. A source of pure water (preferably double distilled water and double distilled water).

 

[ STORAGE OF THE KITS ]

Reagent 1: Precipitant, can be stored at room temperature for 6 month. This solution is supersaturared, if crystals seed out, then take supernatant for experiment.

Reagent 2: Buffer, can be stored at 2～8℃ for 6 months.

Reagent 3: Chromogenic agent, can be stored at 2～8℃ away from light for 6 months.

Reagent 4: GSH standard (Standard powder & Standard solvent stock solution), can be stored at 2～8℃ for 6 months.

 

[ REAGENT PREPARATION ]

GSH standard solvent working solution preparation: When use, dilute standard solvent stock solution with double distilled water at ratio of 1:9, consider solution volume according to your need, working solution should be used soon after preparation.

1mmol/L GSH standard solution preparation: GSH’s molecular weight is 307, add 3.07mg GSH standard powder in 10ml GSH standard solvent working solution, mix sufficiently, can be stored at 2℃～8℃ for 48 hours.

20μmol/L GSH standard solution preparation: Transfer 0.2ml 1mmol/L GSH standard solution in 9.8ml GSH standard solvent working solution. please use this solution soon after preparation.

 

[ SAMPLE PREPARATION ]

1. Cultured cells:

1）Rinse collected cells by PBS 1~2 times, do low speed centrifugation to get cell sediment, add 0.3～0.5ml 0.1M isotonic PBS (pH7.4) to make cell suspension, disrupt cells by ultrasonication or hand-driven.

2）Supernatant preparation: Take cracked cell suspension 0.1ml,and then add 0.1ml Reagent 1,mix well.and then centrifugate at 3500rpm for 10 minutes, take supernatant for assay.

 

2. Tissues:

1）Weigh sample accurately, add 9 times volume physiological saline according to mass (g)-volume(ml) ratio of 1:9, make homogenate in ice water bath, centrifugate at 2500rpm for 10 minutes, take supernatant for assay.

2）Supernatant preparation:Take tissue homogenate 0.1ml,and then add 0.1ml Reagent 1,mix well.and then centrifugate at 3500rpm for 10 minutes, take supernatant for assay.

 

3. Serum、blood plasma:

Take 0.05ml serum (blood plasma), add 0.2ml Reagent 1 working solution, mix sufficiently,  centrifugate at 3500~4000rpm for 10 minutes. Take 1ml supernatant forassay.

 

4. Whole blood：

1）10% hemolysate preparation: Take 0.1ml heparin anticoagulated whole blood, add 0.9ml double distilled water, mix sufficiently until hemolysate becomes limpid.

2）Supernatant preparation: Take 10% hemolysate 0.05ml,and then add 0.2ml Reagent 1,mix well.and then centrifugate at 3500rpm for 10 minutes, take supernatant for assay.

[ ASSAY PROCEDURE ]

	Blank tube	Standard tube	Sample tube
Reagent 1(μl)	100
20μmol/L GSH standard (ml)		100
Supernatant (μl)			100
Reagent 2 (μl)	100	100	100
Reagent 3 (μl)	25	25	25
Mix sufficiently, place for 5 minutes, transferto microplate measure OD values at 405nm.

[ FORMULA ]

1、Whole Blood：

 

2、Cell、Tissue：

 

3、Serum（Blood Plasma）：

 

[ TEST PRINCIPLE ]

Dithio-dinitrobenzoic acid can react with sulfhydryl compounds to produce a yellow compound, it can be quantitative estimated by colorimetric method at 405nm.

 

[ ASSAY SIGNIFICANCE ]

Glutathione (GSH) is a low molecular mundificant which can get rid of O2-, H2O2 & LOOH. GSH is a tripeptide composed by glutamic acid, glycine & cysteine, it is main non-protein sulfhydryl compound in tissue and substrate of GSH-PX & GST (GSH-PX & GST need GSH to decompose hydroperoxides). GSH can also stabilize sulfhydryl containing enzymes and avoid oxidative damage of hemoglobin & other cofactors. Recently, GSH is proved to participate function of recovering vitamin E to reduction form. Absence or exhausting of GSH will induct various chemicals or environmental factors to cause or aggratate toxication, it may relate to increasing oxidative damage, so GSH content is an important factor to measure oxidizing ability in vivo.

 

[STANDARD CURVE]

The sensibility is1.5μmol/L

Dilute 1mmol/L GSH standard solution to：100μmol/L、50μmol/L、20μmol/L、10μmol/L、5μmol/L、0μmol/L to assay for Standard curve.