Description
Detection od Adipose differentiation The adipose tissue works as a place energy store in vivo. The way to do that is turning the energy derived from foods into fat by enzymes has a role in fat synthesis. Among the enzymes, glycerol 3-phosphate dehydrogenase (GPDH) is mostmeasured enzyme that to analyze fat synthesis activity of adipose tissue and adipocyte. This kit has several advantages, in measuring the enzyme activity, over the traditional methods, which include higher stability and reproducibility.
Protocol: 1) Sample preparation - cultured cells
1. Remove culture medium and wash the cells twice with 500 ul PBS.
2. Add enzyme extraction solution to each well. For a 24-well plate, apply 0.5-1 ml per well.
3. Homogenize the cell extract byusing a sonicate.
2)Assay procedure
1. Add 400 ul 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 solutionbecome room temperature. In same way, warm samples at 25°C.
2. Add 200 ul of sample to cuvette and mix it well. Measure decrease of absorbance at 340 nm, and find amount of absorbance change per minute ((DELTA)O.D.). Use kinetic program on spectrometer. If don'thave it, time measurement with timer. In general, measure for 3-10 minutes.
Note 1: As described in example data, find slope ((DELTA)O.D.) on linearity area.
Note 2: It is possible to measure for a maximum of 500 samples, if use 96well micro-platesreader.
3) Calculation of GPDH activity
One unit is activity of 1 umol NADH example by GPDH per minute per 1 ml sample. Based on this, GPDH activity is found following equation. (Only light path is 1 cm )
GPDH activity (U/ ml )=(DELTA)O.D.(340nm )/min. x 0.482##*###
((DELTA)O.D.: value of absorbance change at 340 nm )
##*###96well plate
Light path ( cm ) = total amount of reaction ( ml ) /area of the bottom of well ( cm##2### )
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Protocol: 1) Sample preparation - cultured cells
1. Remove culture medium and wash the cells twice with 500 ul PBS.
2. Add enzyme extraction solution to each well. For a 24-well plate, apply 0.5-1 ml per well.
3. Homogenize the cell extract byusing a sonicate.
2)Assay procedure
1. Add 400 ul 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 solutionbecome room temperature. In same way, warm samples at 25°C.
2. Add 200 ul of sample to cuvette and mix it well. Measure decrease of absorbance at 340 nm, and find amount of absorbance change per minute ((DELTA)O.D.). Use kinetic program on spectrometer. If don'thave it, time measurement with timer. In general, measure for 3-10 minutes.
Note 1: As described in example data, find slope ((DELTA)O.D.) on linearity area.
Note 2: It is possible to measure for a maximum of 500 samples, if use 96well micro-platesreader.
3) Calculation of GPDH activity
One unit is activity of 1 umol NADH example by GPDH per minute per 1 ml sample. Based on this, GPDH activity is found following equation. (Only light path is 1 cm )
GPDH activity (U/ ml )=(DELTA)O.D.(340nm )/min. x 0.482##*###
((DELTA)O.D.: value of absorbance change at 340 nm )
##*###96well plate
Light path ( cm ) = total amount of reaction ( ml ) /area of the bottom of well ( cm##2### )
