Application

This kit can be used to measure G-6-PD activity in animal serum, plasma, tissue, culture cells, bacteria and other samples. 

Detection significance

Glucose-6-phosphate dehydrogenase (G-6-PD) is widely existed in animals, plants, microbes and cultured cells. It is the key enzyme of the phosphopentose pathway. G-6-PD can catalyze the oxidation of 6-phosphate glucose to generate 6- phospho-gluconate, and NADP+ is reduced to NADPH at the same time which can be used for biosynthesis and maintenance of the reduction state in cells. Therefore, the activity of G-6-PD can reflect the biosynthesis and antioxidant capacity of the organism at certain degree. 

Detection principle

G-6-PD catalyze the reduction of NADP+ and produce NADPH, so the G-6-PD activity can be calculated by detecting the increasing rate of OD value of NADPH at 340 nm.

Experimental instrument

Test tube, Mortar, Adjustable micropipettor, Vortex mixer, Centrifuge, Spectrophotometer (340 nm)

Extraction of crude enzyme solution

1. Preparation of bacteria or cells samples:

Collect the bacteria or cultured cells samples into a centrifuge tube, then centrifuge the sample and discard the supernatant. Add Extract Solution into the sediment according to the ratio of Bacteria number / Cells number (104): Extract Solution (mL) =500~1000: 1(it is recommended to add 1 mL of Extract Solution into 2×107 of bacteria or cells), then ultrasonic disrupt the sample (on ice, power: 20% or 200W, 3 seconds/time, interval for 10 seconds, repeat for 30 times). Centrifuge the lysate at 8000g for 10 min at 4℃. Take the supernatant and preserve it on ice for detection.

2. Preparation of tissue samples:

Add Extract Solution into the tissue sample according to the ratio of Weight (g): Extract Solution (mL) =1:5~10 (it is recommended to add 1 mL of Extract Solution into 0.1 g tissue), then homogenize the sample on ice. Centrifuge the tissue homogenate at 8000g for 10 min at 4℃. Take the supernatant and preserve it on ice for detection.

3. Serum / plasma samples:

Detect the serum or plasma samples directly.

Detection procedures

1. Preheat the Spectrophotometer for more than 30 min, then adjust the wavelength at 340 nm and set to zero with distilled water.

2. Add Reagent 2 and Reagent 3 into Reagent 1 and dissolve fully. Incubate the Mixture Reagent at 37℃ (mammal) or 25℃ (other species) for more than 10 min before detection. The unused reagent should be re-pack and stored at -20℃. Avoid repeated freeze-thaw cycles.

3. Add 50 μL of sample and 950 μL of Mixture Reagent into the 1 mL quartz cuvette, mix fully. Measure OD value at 470 nm at 1 min (A1) and 6 min (A2), respectively. △A=A2-A1.

Note: If △A>0.5, dilute the enzyme solution with Extract Solution, and the result should be multiplied by the dilution factor. Or reduce the reaction time to 2 min to make the △A<0.5, which can increase the sensitivity of assay.