Application

This kit can be used to measure T-GSH and GSSG content in serum (plasma), tissue, whole blood, red blood cells and other samples. 

Detection significance

Glutathione is widely distributed in the body. It plays a direct or indirect role in many important biological phenomena, such as mechanism of enzyme reaction, synthesis of biomacromolecules (such as protein and DNA), intermediate metabolism, radiation, tumor, immune, environmental toxins, aging and so on.

Experimental instrument

Test tube, Micropipettor, Vortex mixer, 37℃ water bath/gas bath, Spectrophotometer (412 nm)

Sample pretreatment

1.  Whole blood

(1) Collect blood, use heparin or EDTA as the anticoagulation.

(2) Take 100 μL of whole blood and add 400 μL of reagent 4 working solution, mix fully for 30 s with a vortex mixer, stand for 5 min at 4℃.

(3) Centrifuge at 3500 rpm for 10 min.

(4) Take the supernatant and store at 4℃ for test or -20℃ for over night.

2. Red blood cell

(1) Collect blood, use heparin or EDTA as the anticoagulation.

(2) Centrifuge at 2000 rpm for 10 min immediately, remove the plasma and leukocytic layer (upper layer) carefully.

(3) Take 100 μL of red blood cell, add 400 μL of reagent 4 working solution, mix fully for 30 s with a vortex mixer, stand for 5 min at 4℃.

(4) Take the supernatant and store at 4℃ for test or -20℃ for over night.

3. Serum/plasma

(1) Prepare serum/plasma as the common method.

(2) Take 100 μL of sample and add 400 μL of reagent 4 working solution to it, mix by a vortex mixer for 30 s, let it stand at 4℃ for 5 min.

(3) Centrifuge at 3500 rpm for 10 min.

(4) Take the supernatant and store at 4℃ for test or -20℃ for over night.

4. Tissue homogenate

(1) Collect fresh tissue, wash with normal saline, then absorb the water on surface of the tissue.

(2)  Weigh the tissue accurately, adding reagent 4 working solution according to ratio of Weight (g): Volume (mL) =1:4. Homogenize mechanically with a homogenizer in ice-bath to prepare 20% homogenate.

(3) Centrifuge at 3500 rpm for 10 min.

(4) Take the supernatant and store at 4℃ for test or -20℃ for over night.

Operation Steps

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment. Dilute the sample with reagent 4 working solution if the absorbance exceed the linear range.

1. Detection of T-GSH

Record the time after adding reagent 3 working solution, mix fully, measure the absorbance at 412 nm with 1 cm diameter cuvette at 30 second (A1) and 630 second (A2), respectively. △A=A2-A1.

2. Detection of GSSG

(1) Pretreatment

   Mix fully for 1 min, react for 30 min at 37℃, then take 50 μL for test.

(2) Determination of GSSG content

    Record the time after adding reagent 3 working solution, mix fully, measure the absorbance at 412 nm with 1 cm diameter cuvette at 30 second (A1) and 630 second (A2), respectively. △A=A2-A1.

Notes

1. This product is for scientific research use only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 3 months.

4. Do not use components from different batches of kit.

5. It’s better treat samples with reagent 4 working solution as soon as possible to reduce the loss of GSH.

6. Samples treated with reagent 4 working solution can be stores at -20℃ for 6 months.

7. The reaction time is 10 min which must be record accurately.