Glutathione S-Transferase Assay Kit

Instruction manual

First Edition (Revised on April, 2016)

[ INTENDED USE ]

This kit applies to measure Glutathione S-Transferase (GSH-ST) activities in blood serum, tissues, cultured cells, etc.

[ REAGENTS AND MATERIALS PROVIDED ]

Reagents		Quantity(100T-48S)	Reagents		Quantity(100T-48S)
Reagent 1	Powder A	1	Reagent 2	Powder A	1
	Solution B	1×60ml		Solution B	1×50ml
Reagent 3		1	Reagent 4		1
Reagent 5		3	Reagent 6		1×10ml
Instruction manual		1

[ MATERIALS REQUIRED BUT NOT SUPPLIED ]

1. An spectrophotometer capable of measuring absorbance at 412nm.

2. Thermostatic water bath or air bath capable of controlling temperature at 37℃. 

3. Low speed centrifuge.

 

[ STORAGE OF THE KITS ]

1. This kit can be stored at 2-8℃ hermetically for 6 months.

2. Reagent 1:Powder A and Solution B, can be stored at 2～8℃ temperature for 6 months.

3. Reagent 2:Powder A and Solution B, can be stored at 2～8℃ temperature for 6 months.

4. Reagent 3: White Powder, can be stored a 2～8℃ for 6 months.

5. Reagent 4:Light yellow powder, can be stored at 2～8℃ for 6 months.

6. Reagent 5: GSH standard powder, 3.07mg×3 vials, can be stored at 2～8℃ for 6 months.

7. Reagent 6:GSH standard solvent stock solution, can be stored at 2～8℃ for 6 months.

 

[ REAGENT PREPARATION ]

Reagent 1 - Solution A preparation: When use, add 1ml dehydrated alcohol to dissolve powder A(Reagent 1-powder A) completely, can be stored at 2～8℃.

Reagent 1 working solution preparation: Mix solution A(Reagent 1-powder A) and solution B(Reagent 1-powder B) at volume ratio of 1:59, working solution should be used soon after preparation.

Reagent 2 working solution preparation: Dissolve powder A(Reagent 2-powder A) completely in 170ml hot double distilled water at 90~100℃ to make solution A(Reagent 2 - Solution A), mix solution A(Reagent 2 - Solution A) and solution B(Reagent 2 - Solution B) sufficiently. Working solution can be stored at room temperature, it is supersaturated, if crystals seed out, then take supenatant for assay.

Reagent 3 working solution preparation：Add 200ml double distilled water to dissolve Reagent 3 completely, can be stored in plastic bottle (given by Cloud-clone) at room temperature.

Reagent 4 working solution preparation: Add 50ml double distilled water to dissolve Reagent 4 completely, can be stored at 2～8℃ away from light.

GSH standard solvent working solution preparation: Dilute GSH standard solvent stock solution with double distilled water at volume ratio of 1:9 (equals to 10 times dilution).       

1mmol/L GSH standard solution preparation: Take 1 vial GSH standard (3.07mg), dilute with 10ml prepared standard solvent working solution, mix sufficiently to dissolve completely.

20mmol/L standard solution preparation: Dilute 0.2ml GSH standard solution with 9.8ml GSH standard solvent working solution.

Matrix buffer preparation: Mix Reagent 1 working solution and 1mmol/L GSH standard solvent at ratio of 1:1.

[ SAMPLE PRETREATMENT ]

1. 50 times diluted hemolysate preparation: Take 20ml heparin anticogulated whole blood, add double distilled water until volume reaches 1ml, mix sufficiently, place for 5 minutes then start assay. Prepared hemolysate should be measured in 1 hour or activity will be disturbed. Anticogulated whole blood can be stored at 4℃, enzyme activity keeps stable in 2~3 days.

2. Preparation of tissue homogenate, mitochondria, microsome should follow experimental methodology.

3. Blood serum or plasma sample can be take directly.

[ OPERATION PROCEDURE ]

1. Blood serum (or plasma) GST activity assay:

① Enzymatic reaction:

	Sample tube	Contrast tube
Matrix buffer (ml)	0.3	0.3
Blood serum（or plasma）(ml)	0.1
Mix sufficiently, place in 37℃ water bath for 30 minutes
Reagent 2 working solution (ml)	2	2
Blood serum（or plasma）(ml)		0.1
Mix sufficiently, centrifugate 3500~4000rpm for 10 minutes, take supernatant for chromogenic reaction

② Chromogenic reaction:

	Blank tube	Standard tube	Sample tube	Contrast tube
GSH standard solvent working solution (ml)	2
20mmol/LGSH standard solution (ml)		2
Supernatant (ml)			2	2
Reagent 3 working solution (ml)	2	2	2	2
Reagent 4 working solution (ml)	0.5	0.5	0.5	0.5
Mix sufficiently, place at room temperature for 15 minutes, transfer in cuvette of 25px light path, measure OD values at 412nm (adjust zero by double distilled water).

③ Calculation:

Unit definition: 1ml blood serum (or plasma) reacts at 37℃ for 1 minute, subtract non-enzymatic reaction, 1mmol/L GSH concentration reducing in reaction system is considered as 1 enzyme activity unit (U).

Formula:

 

Note: OD value equals to absorbance.

 

④ Example:

a. Take 0.1ml Tilapia blood serum to measure GSH-ST activity, in results, OD_Sample is 0.436, OD_Contrast is 0.485, OD_Standard is 0.160, OD_Blank is 0.037, calculate as follows:

b. Take 0.1ml swine blood serum to measure GSH-ST activity, in results, OD_Sample is 0.403, OD_Contrast is 0.475, OD_Standard is 0.160, OD_Blank is 0.037, calculate as follows:

 

2. Tissue GST activity assay:

① Enzymatic reaction:

Homogenate supernatant (ml)

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Mix sufficiently, centrifugate 3500~4000rpm for 10 minutes, take supernatant for chromogenic reaction

② Chromogenic reaction:

	Blank tube	Standard tube	Sample tube	Contrast tube
GSH standard solvent working solution (ml)	2
20mmol/LGSH standard solution (ml)		2
Supernatant (ml)			2	2
Reagent 3 working solution (ml)	2	2	2	2
Reagent 4 working solution (ml)	0.5	0.5	0.5	0.5
Mix sufficiently, place at room temperature for 15 minutes, transfer in cuvette of 25px light path, measure OD values at 412nm (adjust zero by double distilled water).

③ Calculation:

Unit definition: 1mg tissue protein reacts at 37℃ for 1 minute, subtract non-enzymatic reaction, 1mmol/L GSH concentration reducing in reaction system is considered as 1 enzyme activity unit (U).

Formula:

Note: OD value equals to absorbance, mgprot/ml means milligram protein per ml.

 

④ Example:

a. Take 0.1ml 10% rat brain tissue homogenate to measure GSH-ST activity, in results, OD_Sample is 0.218, OD_Contrast is 0.462, OD_Standard is 0.160, OD_Blank is 0.037, protein concentration is 4.15mgprot/ml, calculate as follows:

b. Take 0.1ml 10% squid brain tissue homogenate to measure GSH-ST activity, in results, OD_Sample is 0.228, OD_Contrast is 0.460, OD_Standard is 0.160, OD_Blank is 0.037, protein concentration is 3.57mgprot/ml, calculate as follows:

 

3. 50 times diluted hemolysate GST activity assay:

① Enzymatic reaction:

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Sample tube	Contrast tube
Matrix buffer (ml)	0.2	0.2
Homogenate supernatant (ml)	0.2
Mix sufficiently, place in 37℃ water bath for 30 minutes.
Reagent 2 working solution (ml)	2	2
Homogenate supernatant (ml)		0.2
Mix sufficiently, centrifugate 3500~4000rpm for 10 minutes, take supernatant for chromogenic reaction.

② Chromogenic reaction:

	Blank tube	Standard tube	Sample tube	Contrast tube
GSH standard solvent working solution (ml)	2
20mmol/LGSH standard solution (ml)		2
Supernatant (ml)			2	2
Reagent 3 working solution (ml)	2	2	2	2
Reagent 4 working solution (ml)	0.5	0.5	0.5	0.5
Mix sufficiently, place at room temperature for 15 minutes, transfer in cuvette of 25px light path, measure OD values at 412nm (adjust zero by double distilled water).

③ Calculation:

Unit definition: 1ml whole blood reacts at 37℃ for 1 minute, subtract non-enzymatic reaction, 1mmol/L GSH concentration reducing in reaction system is considered as 1 enzyme activity unit (U).

Formula:

Note: OD value equals to absorbance, mgprot/ml means milligram protein per ml, 0.2ml 50 times diluted hemolysate equals ti 0.004ml whole blood (0.2ml÷50=0.004ml).

 

④ Example:

Use 20ml whole blood to make 1:49 hemolysate, take 0.2ml hemolysate to measure GSH-ST activity, in results, OD_Sample is 0.231, OD_Contrast is 0.280, OD_Standard is 0.160, OD_Blank is 0.037, protein concentration is 4.15mgprot/ml, calculate as follows:

 

[ ASSAY SIGNIFICANCE ] 

Glutathione S-transferase（GSH-ST or GST）is a liver detoxication related enzyme. Liver cells contain large amount of GST, as result, when liver cells are damaged, GST always releases into blood at very early stage. In blood, GST increases always earlier than glutamate pyruvate transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT), so GST increasing can be used as sensitive factor for liver damage.

Various mammal tissues contain GST, which can catalyze glutathione reacts with electrophilic group to produce mercapturic acid and discharge in vitro, it plays an important role in detoxication in vivo.

GST has functions both of eliminate peroxides in vivo and detoxication. But if glutathione peroxidase (GSH-PX) activity is low, then GST only has function to eliminate lipid peroxides (LPO) in vivo.

 

[ ASSAY PRINCIPLE ]

GST is able to catalyze reduced glutathione (GSH) combined with 1-chloro-2,4-dinitrobenzene (CDNB), in a certain reaction period, its activity appears linear correlation with substrate concentration variance before and after reaction.

This kit uses GSH concentration to reflect GST activity, larger GSH concentration reducing relates to higher GST activity.

 

 

 

[ANNOUNCEMENTS]

1. All test tubes should be cleansed, please brush test tubes with warm soapsuds at first, then rinse test tubes by running water at least 20 times, finally, you can wash test tubes with distilled water 1~2 times then dry them by baking.

2. Reagent 2 should be dissolved completely at hot temperature (90℃~100℃).

3. Heparin anticoagulated whole blood can be stored in fridge less than 3 days, blood plasma and tissue pieces can be stored at -20℃ for more than 1 month.

4. Reagent 1 stock solution can be stored in fridge more than 1 month.

5. 1mmol/L GSH substrate should be used soon after preparation.

5. Supernatants should be prepared and used in the same day.

6. There are several methods to measure tissue protein content, you can buy assay kits from our institute.

7. Sample pretreatments should follow Experimental Methodology.

8. This kit can be used in scientific research and laboratory only.