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Glycerol-3-Phosphate Dehydrogenase Activity Colorimetric Assay Kit

Highly-sensitive assay to measure GPDH activity
Catalog #: K640

In stock

$515.00

Product Details

Cat # +Size K640-100
Size 100 assays
Detection Method Absorbance (450 nm)
Applications Measurement of Glycerol-3-Phosphate Dehydrogenase activity in various tissues/cells
Analysis of fatty acid metabolism and cell signaling
Screening anti-obesity drugs
Features & Benefits • Simple, sensitive, and rapid.
• The assay can detect Glycerol-3-Phosphate dehydrogenase activity less than 1 mU/well.
Kit Components • GPDH Assy Buffer
• GPDH Substrate (Lyophilized)
• GPDH Probe (Lyophilized)
• NADH Standard (Lyophilized)
• GPDH Positive Control (Lyophilized)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

Glycerol-3-Phosphate Dehydrogenase (EC 1.1.1.8) is an important enzyme for lipid metabolism. It catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-3-phosphate. GPDH plays multiple functions inside cells; it links carbohydrate and lipid metabolism, and provides electrons through the Glycerol-3-Phosphate Shuttle. When progenitor adipocytes differentiate into mature adipocytes, GPDH activity increases significantly. Analysis of glycerol-3-phosphate dehydrogenase activity is crucial for the study of fatty acid metabolic pathways. In BioVision’s Glycerol-3-Phosphate Dehydrogenase Activity Assay kit, Glycerol-3-phosphate dehydrogenase forms an intermediate, which reduces a colorless probe to a colored product with strong absorbance at 450 nm. The assay is simple, sensitive and rapid and can detect Glycerol-3-Phosphate dehydrogenase activity less than 1 mU/well.


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Does this kit measure cytoplasmic G3PDH only?
This kit is based on function of the G6PDH enzyme. You can use cytosolic fraction or whole cell/tissue lysate to quantify amount of active G6PDH enzyme.
Can this kit also measure glycerol in the sample?
No, this kit is to measure activity of the GPDH enzyme. If you wish to measure glycerol please use K630 instead.
Can the assay buffer used to grow cells? Is it similar to WME?
The Assay buffer is optimized for the detection reaction (pH, salts etc optimized for the enzymes in the enzyme mix to work). This is not like Williams medium E for culture of cells.
How many cells should be grown per well so that the medium can then be assayed for lactate?
There are several variables : 1) Cell type and the metabolic sate of the cell 2) Medium glucose level 3) lactate/glucose ratio in the media. Since cell types vary, the number of cells used depends on the cell type and experimental data needs to be generated to predict the level of lactate Vs. time. As time passes, cells will be consuming glucose and produce lactate. As the level of glucose drops and the cell starve, they start consuming lactate. So, the level of lactates goes up with time and eventually it starts decreasing.
Can this assay be normalized with respect to protein concentration?
Yes, a detergent-compatible BCA assay can be used for protein quantitation to normalize sample amount.
Can frozen samples be used with this assay?
Fresh samples are always preferred over frozen samples since this is an enzyme activity assay. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Is it possible to use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. It is recommended to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Can individual components of this kit be purchased separately?
Yes, any of the kit's components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Is it essential to make a standard curve for every expt, or is one curve/kit enough?
Yes, it is strongly recommended to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
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