• HOME
  • Products
  • GPDH [glycerol 3 -phosphate dehydrogenase] Assay Kit

For research of fat differentiation

GPDH [glycerol 3 -phosphate dehydrogenase] Assay Kit


print

Background

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 340nm by spectrophotometry.

Kit component

Component Quantity Form Stability
SUBSTRATE 10 Bottles
(1 bottle=10 tests)
Freeze Dry -20°C
ENZYME EXTRACTION REAGENT 1 Bag Powder -20°C

One kit contains reagents for 100 samples

Protocol

Sample preparation - Cultured cells

1.  Remove culture medium and wash the cells twice with 500μl 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

Assay procedure
1.  Add 400μL 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 200μL of sample to cuvette and mix it well. Measure decrease of absorbance at 340nm, and find amount of absorbance change per minute (?O.D.). Use kinetic program on spectrometer. If don’t have it, time measurement with timer. In general, measure for 3~10minutes.

Note 1: As described in example data, find slope (?O.D.) on linearity area.
Note 2: It is possible to measure for a maximum of 500 samples, if use 96well micro-plates reader.

Calculation of GPDH activity
One unit is activity of 1μmol 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) = ?O.D.(340nm)/min ׁ@0.482 *
(?O.D. : value of absorbance change at 340nm)

*96well plate
Light path (cm) = total amount of reaction (mL) /area of the bottom of well (cm2)

Application example in primary culture cell
from adipose tissue or cell lines (i.e.:3T3-L1, 3T3-f442, ob1771)

Example Data

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


References

> Tashiro, K., Inamura, M., Kawabata, K., Sakurai, F., Yamanishi, K., Hayakawa, T., Mizuguchi, H. Efficient Adipocyte and Osteoblast Differentiation from Mouse Induced Pluripotent Stem Cells by Adenoviral Transduction. Stem Cells. 27, 1802-1811 (2009)
> Nagane, K., Jo, J., Tabata, Y. Promoted Adipogenesis of Rat Mesenchymal Stem Cells by Transfection of Small Interfering RNA Complexed with a Cationized Dextran. Tissue Eng. Part A. 16, 21-31(2010)
> Matsumura, K., Bae,JY., Hyon SH. Polyampholytes as Cryoprotective Agents for Mammalian Cell Cryopreservation. Cell Transplant. 19, 691-699 (2010)
> Jiao, WH., Gao, H., Li, CY., Zhou, GX., Kitanaka, S., Ohmurae, A., Yao, XS. beta-Carboline Alkaloids from The Stems of Picrasma quassioides. Magn. Reson. Chem. 48, 490-495 (2010)

Product List

Product Name Cat# Quantity Price

GPDH Assay Kit DataSheet ,

PMC-AK01-COS

1 KIT ¥60,000
$800

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.



“GPDH [glycerol 3 -phosphate dehydrogenase] Assay Kit” also belongs to the following categories.