GPDH Assay Kit

Catalog No. ABIN955779

Product Details

Target: GPD2 (Glycerol Phosphate Dehydrogenase 2, Mitochondrial (GPD2))

Sample Type: Cell Culture Samples, Tissue Homogenate

Components: 10 x Substrate Reagent, Lyophilized, contains DHAP and NADH
1 x Enzyme Extracting Reagent, Lyophilized
1 kit = 100 tests (when using a 96-well microplate, up to 500 tests can be run).

Material not included: Purified water
Adjustable pipettor
PBS
Spectrophotometer or microplate reader capable of measuring at 340 nm
Quartz cuvette for spectrophotometer method or microtiter plate for microplate method
Centrifuge
Centrifuge tubes
Sonicator.

Application Details

Sample Preparation:

1. Tissue Sample:
   a. Add 4 mL of 0.25M sucrose solution to 1 gram of adipose or other tissue and homogenize the mixture.
   b. Centrifuge at 700 x g at 4°C for 10 minutes. Remove the supernatant (do not discard).
   c. Centrifuge the supernatant at 54,000 x g at 4°C for 60 minutes. Remove the supernatant (cytosol fraction).
   d. Dilute the supernatant approximately 20 to 100 times with reconstituted Enzyme Extracting Reagent.
   e. Assay the sample.
   
   2. Cultured Cells:
   a. Remove culture medium and wash sample cells twice with PBS.
   b. Add the reconstituted Enzyme Extracting Reagent to the sample.
   c. When a 24-well plate is used, add the reconstituted Enzyme Extracting Reagent at 0.5 to 1.0 mL per well.
   d. Scrape cells with a sterile rubber policeman. Transfer cells to a clean centrifuge tube.
   e. Use a sonicator to homogenize the cell extracts.
   f. Crude extracts may be directly assayed or centrifuged at 12,800 x g at 4°C for 5 minutes. Centrifugation is recommended.
   g. Use the supernatant as the sample for the assay.

Restrictions: For Research Use only

Handling

Handling Advice: 1. Read the instructions carefully before beginning the assay.
2. This kit is for research use only, not for human or diagnostic use.
3. Great care has been taken to ensure the quality and reliability of this product. However, it is possible that in certain cases, unusual results may be obtained due to high levels of interfering factors.

Storage: -20 °C

Storage Comment: Kit components can be stored at -20°C until expiration date. Upon reconstitution, store the Enzyme Extracting Reagent at 4°C for up to 4 weeks and the Substrate Reagent at 4°C for up to 2 days.

Expiry Date: The expiry date is stated on the label.

Target Details

Target: GPD2 (Glycerol Phosphate Dehydrogenase 2, Mitochondrial (GPD2))

Alternative Name: GPDH (GPD2 Products)

Synonyms: glycerol-3-phosphate dehydrogenase Gpd2 Kit, gpd2 Kit

Background: An organism's major sources of fatty acids come from its diet or mobilization from cellular storage. Fatty acids from the diet are solubilized and absorbed through the gut and delivered to the cells via blood transport. Excess free state long chain fatty acids are cytotoxic in cells. Adipocytes avoid the accumulation of fatty acids by storing it in the form of triacylglycerols. In adipose tissue, GPDH reduces dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate using coenzyme NAD. The sequential binding of three glycerol 3-phosphates by coenzyme acyl-CoA generates triacylglycerol. In response to energy demands, the fatty acids stored as triacylglycerols can be utilized by peripheral tissues.The measurement of GPDH activity is widely used to assess the biosynthesis of fat in adipocytes and adipose tissues. The activity of GPDH rapidly increases upon differentiation of precursor adipocytes to mature adipocytes. The GPDH Activity Measurement Kit offers a simple and rapid assay procedure for assaying tissue samples or cultured cells for GPDH. When DHAP (dihydroxyacetone phosphate) and NADH are mixed in the presence of GPDH from the sample, glycerol-3-phoshate and NAD are produced. The decrease in NADH concentration is then measured at 340 nm. GPDH DHAP + NADH Glycerol-3-phosphate + NAD.