Application

This kit can be used to measure α-Ketoglutarate Dehydrogenase (α-KGDH) activity in tissue, culture cells, bacteria, mitochondria and other samples. 

Detection significance

α-KGDH is widespread in mitochondria of animals, plants, microorganisms and cultured cells. It is a key enzyme in the regulation of tricarboxylic acid cycle, which catalyzes the oxidation of α -ketoglutaric acid to produce succinylcoenzyme A.

Detection principle

α-KGDH catalyzes α- ketoglutaric acid, NAD+ and coenzyme A to produce succinyl coenzyme A, carbon dioxide and NADH. NADH has a characteristic absorption peaks at 340nm. The activity of α -KGDH can be calculated by measuring the production rate of NADH.

Experimental instrument

Micro quartz cuvette / 96 wells Micro-plate, High-precision transferpettor, Vortex mixer, Centrifuge, Spectrophotometer (340 nm) / Microplate Reader (340 nm), Water bath, Mortar, distilled water

Pretreatment of sample

The separation of cytoplasmic and mitochondrial proteins from tissues, bacteria, or cells

1. Weigh 0.1 g tissue or collect 5×106 cells, add 1 mL of reagent 1 and 10 μL reagent 3, homogenize the sample in ice bath.

2. Centrifuge at 600 g for 5 min at 4℃.

3. Discard the sediment, take the supernatant to another centrifuge tube. Centrifuge at 11000 g for 10 min at 4℃.

4. (Optional step)The supernatant is the cytoplasmic extract. It can be used for the determination of α-KGDH leaking from mitochondria.

5. Take the sediment in step 4, add 200 μL of reagent 2 and 2 μL of reagent 3, then treat the sample with sonication on ice (power: 20% or 200W, 3 seconds/time, the interval for 10 seconds, repeat for 30 times). Take the supernatant and preserve it on ice for detection.

Detection procedures

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

2. Incubate the reagent 5 application solution at 37℃ (for mammal samples) or 25℃ (for other species samples) for more than 10 min before detection.

3. Add 10 μL of sample, 10 μL of reagent 6 application solution and 180 μL of reagent 5 application solution into the micro quartz cuvette or 96 wells Microplate, mix fully. Measure the absorbance at 340 nm at 20 s (A1) and 140 s (A2), respectively. Calculate the △A=A2-A1.

Notes

1. The kit is for scientific research only.

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

3. The valid of kit is 3 months.

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

5. If the activity of α-KGDH is calculated by protein concentration, the protein concentration of the sample needs to be determined separately (E-BC-K318, E-BC-K168, E-BC-K165).