Adipose tissue functions as a place to store energy in vivo. The way to store energy is to convert the energy derived from foods into fat by enzymes which have a role in fat synthesis. Among the various enzymes, glycerol 3 phosphate dehydrogenase (GPDH) is the most measured enzyme to analyze fat synthesis activity of adipose tissue and adipocytes.
This kit has several advantages - a superb measuring enzyme activity over traditional methods, which includes higher stability and reproducibility of results.
|Dihydroxyacetone-phosphate + NADH →→→ Glycerol-3-phosphate + NAD
The decrease of NADH is measured at 340 nm by spectrophotometry.
(1 bottle=10 tests)
|ENZYME EXTRACTION REAGENT||1 Bag||Powder||-20°C|
One kit contains reagents for 100 samples
Sample preparation - Cultured cells
1. Remove culture medium and wash the cells twice with 500 μL PBS.
2. Add enzyme extraction solution to each well. For a 24-well plate, apply 0.5~1mL per well.
3. Homogenize the cell extract by using a sonicato
1. Add 400 μL of substrate solution to spectrometer cuvette (quarts micro cuvette), and warm at 25°C (About 5 minutes). Hopefully use spectrometer keep warm at 25°C. When couldn’t, wait until substrate solution become room temperature. In same way, warm samples at 25°C.
2. Add 200 μL of sample to cuvette and mix it well. Measure decrease of absorbance at 340 nm, and find amount of absorbance change per minute (ΔO.D.). Use kinetic program on spectrometer. If don’t have it, time measurement with timer. In general, measure for 3~10 minutes.
Note 1: As described in example data, find slope (ΔO.D.) on linearity area.
Note 2: It is possible to measure for a maximum of 500 samples, if use 96-well micro-plates reader.
Calculation of GPDH activity
One unit is activity of 1 μmol NADH example by GPDH per minute per 1mL sample. Based on this, GPDH activity is found following equation. (Only light path is 1cm)
GPDH activity (U/mL) = ΔO.D.(340 nm)/min × 0.482 *
(ΔO.D. : value of absorbance change at 340 nm)
Light path (cm) = total amount of reaction (mL) /area of the bottom of well (cm2)
Effect of Troglitazone on GPDH activity in 3T3-L1 cell cultures
GPDH activities were measured with GPDH Assay Kit.
A, 3T3-L1 adipocytes treated with Troglitazone
B, 3T3-L1 adipocytes
C, 3T3-L1 preadipocytes
GPDH Assay Kit
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.