Application
This kit can be used to measure the GSH-ST activity in animal serum, plasma, tissue, Whole blood, cell and other samples.
Detection significance
Glutathione S- transferase (GSH-ST) is a kind of enzyme related to liver detoxification, which abounds in liver cells. When liver cells are damaged, increase of GSH-ST in blood often appears earlier than glutamic-pyruvic transaminase (SGPT) and glutamic oxalacetic transaminase (SGOT), so the increase of GSH-ST can be regarded as a sensitive index of liver injury.
Detection principle
GST can catalyze the binding of reduced glutathione (GSH) to 1-chlorine-2, 4-dinitrobenzene. In a certain reaction time, the activity of GST is linearly related to the change of substrate concentration after the reaction. This kit calculate the activity of GST by detecting the concentration of GSH.
Experiment instruments
Test tube,
Micropipettor,
Vortex mixer,
Centrifuge,
37℃ water bath,
Spectrophotometer (412 nm)
Sample preparation
1. Serum/plasma: Detect the sample directly. If the concentration is beyond the linear range, dilute the sample with saline before detection.
2. Whole blood:
Preparation of hemalysates:
(1) Take 20 μL of whole blood anticoagulated with heparin. Dilute the sample with double-distilled water to 1 mL to prepare 1:49 hemolysis. For mouse/rat blood, dilute 10 μL of blood with double-distilled water to 1 mL to prepare 1:99 hemalysates.
(2) Mix fully and stand for 5 min until the hemalysates totally transparent for light, then operate the detection.
(3) The GSH-ST activity of prepared hemalysates only maintains for 45~60 min, and it can last for 120 min under cold condition. The anticoagulated whole blood can be stored at 4℃ for 2~3 days.
3. Tissue sample:
(1) Take the tissue (0.2~1 g) and wash with cooling normal saline. Remove the blood and dry with filter paper. Weigh the tissue and put it into a 5 mL/10 mL beaker.
(2) Pipette homogenization medium (pH7-7.4, 0.01 mol/L sucrose, 0.01 mol/L Tris-HCl, 0.0001 mol/L EDTA·2Na) or 0.86% normal saline with a ratio of Weight (g): Volume (mL) =1:9. Transfer 2/3 of the total homogenate medium or normal saline to the beaker. Mince the tissue into small pieces with ophthalmic scissors.
(3) Add the tissue pieces into a glass homogenate tube, wash the beaker with the other 1/3 homogenate medium or 0.86% normal saline to collect the residual tissue into the tube. Mechanical homogenate the sample in ice water bath.
(4) Centrifuge the prepared 10% tissue homogenate at 3000 r/min for 10~15 min. Then take the supernatant for detection.
4. Preparation of mitochondria:
Take 5~10 mL of 10% tissue homogenate and centrifuge at 1000~2000 r/min for 10 min. Then centrifuge the supernatant at 8000~10000 r/min for 15 min (low-temperature and high-speed centrifuge). The sediment is mitochondria.
5. Cells and culture supernatant sample:
Cell sample: Digest the cell sample and centrifuge at 1000~1500 r/min for 10 min. Remove the supernatant and keep the cell sediment. Add 0.3~0.5 mL of normal saline or homogenate medium into each tube to prepare 106/mL cell suspension. Sonicate in ice water bath (power: 300 W, 3~5 sec/time, interval for 30 sec, repeat for 3~5 times) or grind with hand-operated. The prepared homogenate liquid kept for detection without centrifugation.
Culture supernatant: Centrifuge at 1000~1500 r/min for 10 min. Collect the supernatant for detection.
Operation procedure
1. For serum(plasma) sample
a) Enzymatic reaction
| Sample tube | Contrast tube |
Substrate solution (mL) | 0.3 | 0.3 |
Sample (mL) | 0.1 |
|
Mix fully and incubate for 30 minutes in 37℃ water bath accurately. | ||
Reagent 2 application solution (mL) | 2 | 2 |
Sample (mL) |
| 0.1 |
Mix fully, centrifuge at 3500~4000 r/min for 10 min. Collect the supernatant for chromogenic reation.
b) Chromogenic reaction
| Blank tube | Standard tube | Sample tube | Contrast tube |
GSH Standard application solution (mL) | 2 |
|
|
|
20 μmol/L GSH standard solution (mL) |
| 2 |
|
|
Supernatant (mL) |
|
| 2 | 2 |
Reagent 3 application solution (mL) | 2 | 2 | 2 | 2 |
Reagent 4 application solution (mL) | 0.5 | 0.5 | 0.5 | 0.5 |
Mix fully and stand for 15 min at room temperature. Set to zero with double-distilled water. Measure the OD values of each tube at 412 nm with 1 cm diameter cuvette.
2. For tissue sample
a) Enzymatic reaction
Mix fully, centrifuge at 3500~4000 r/min for 10 min. Collect the supernatant for chromogenic reation.
b) Chromogenic reaction
| Blank tube | Standard tube | Sample tube | Contrast tube |
GSH Standard application solution (mL) | 2 |
|
|
|
20 μmol/L GSH standard solution (mL) |
| 2 |
|
|
Supernatant (mL) |
|
| 2 | 2 |
Reagent 3 application solution (mL) | 2 | 2 | 2 | 2 |
Reagent 4 application solution (mL) | 0.5 | 0.5 | 0.5 | 0.5 |
Mix fully and stand for 15 min at room temperature. Set to zero with double-distilled water. Measure the OD values of each tube at 412 nm with 1 cm diameter cuvette.
3. For whole blood sample
a) Enzymatic reaction
| Sample tube | Contrast tube |
Substrate solution (mL) | 0.2 | 0.2 |
Sample (mL) | 0.2 |
|
Mix fully and incubate for 30 minutes in 37℃ water bath (time accurately). | ||
Reagent 2 application solution (mL) | 2 | 2 |
Sample (mL) |
| 0.2 |
Mix fully, centrifuge at 3500~4000 r/min for 10 min. Collect the supernatant for chromogenic reation.
b) Chromogenic reaction
| Blank tube | Standard tube | Sample tube | Contrast tube |
GSH Standard application solution (mL) | 2 |
|
|
|
20 μmol/L GSH standard solution (mL) |
| 2 |
|
|
Supernatant (mL) |
|
| 2 | 2 |
Reagent 3 application solution (mL) | 2 | 2 | 2 | 2 |
Reagent 4 application solution (mL) | 0.5 | 0.5 | 0.5 | 0.5 |
Mix fully and stand for 15 min at room temperature.
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