Product Details
| abID | ab183305 |
|---|---|
| Cat # +Size | K654-100 |
| Size | 100 assays |
| Detection Method | Absorbance (450 nm) |
| Applications | Measurement of malate dehydrogenase activity in various tissues/cells Analysis of citric acid cycle and malate-asparate shuttle |
| Features & Benefits | • Simple, rapid & convenient • The assay can detect Malate Dehydrogenase (MDH) Activity less than 0.5 mU in various sample types. |
| Kit Components | • MDH Assay Buffer • MDH Substrate (Lyophilized) • MDH Enzyme Mix (Lyophilized) • MDH Developer (Lyophilized) • NADH Standard (Lyophilized) • MDH Positive Control (Lyophilized) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Malate Dehydrogenase (MDH) (EC 1.1.1.37) is an important enzyme which reversibly converts L-malate into oxaloacetate in the presence of NAD. In eukaryotic cells, malate dehydrogenase has 2 isoforms: MDH1 and MDH2. MDH1 is cytosolic & participates in the malate-aspartate shuttle, which transports malate into mitochondria for utilization in ATP generation whereas MDH2 is a mitochondrial enzyme and part of the citric acid cycle. MDH activity is increased in some neurodegenerative diseases such as Alzheimer’s disease, and abnormal MDH activity in serum can serve as a diagnostic tool for severe liver damage (e.g. Hepatocellular carcinoma). In BioVision’s Malate Dehydrogenase Activity Assay kit, MDH reacts with malate to form an intermediate. The generated intermediate reacts with MDH Developer to form a colored product with strong absorbance at 450 nm. The assay is simple, sensitive and can detect less than 0.5 mU of MDH activity in various sample types.
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Does it rely on an immunoprecipitation step to isolate MDH from tissues? Also, is it possible to use purified protein for this assay? If so, is there a recommended working range?
No, there is no immunopreceipitation done in this assay. Purified MDH enzyme can be used . The readings need to be within the linear range of the std curve. How much sample is used needs to be optimized to achieve this. This kit can detect from 0.1 -2.5 mU of enzyme activity.
Can mitochondrial MDH activity be measured?
Mitochondria isolated from the tissue/cell sample of interest and then solubilized using the assay buffer can be used to measure mitochondrial MDH specifically.
Does the MDH positive control consist of MDH1 protein, MDH2 protein, or both?
The positive control is from mitochondria (MDH2).
Which isoform of MDH is meassure by this kit?
It depends on which sub-cellular fraction is used for the assay. Total cell lysate will measure both MDH 1 and 2., cytosolic fraction will be from MDH1 and mitochondria will have MDH2.
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 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.
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.
Can cells lysed in RIPA buffer be used with this kit?
RIPA buffer typically contains SDS which might affect the function of the enzymes in the kit. Hence we do not recommend using RIPA bufefr samples. The assay buffer in the kit also provides optimum conditions for the enzymes to work at their best.
López-Ibarra et al, Metabolic differences between white and brown fat from fasting rabbits at physiological temperature.J. Mol. Endocrinol., Feb 2015; 54: 105 - 113.
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