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Alpha-Ketoglutarate Dehydrogenase Activity Colorimetric Assay Kit

based on 1 citations in multiple journalsAlpha-Ketoglutarate Dehydrogenase Activity Colorimetric Assay Kit14.1 4
Catalog #: K678

In stock

$655.00

Product Details

Cat # +Size K678-100
Size 100 assays
Detection Method Absorbance (450 nm)
Applications Measurement of α-Ketoglutarate dehydrogenase activity in various tissues/cells
Analysis of cell signaling pathways such as citrate acid cycle, lysine degradation or tryptophan metabolism in various cell types
Features & Benefits • Simple, rapid & convenient
• can measure α-ketoglutarate dehydrogenase activity lower than 0.1 mU in a variety of samples
Kit Components • KGDH Assay Buffer
• KGDH Substrate (Lyophilized)
• KGDH Developer (Lyophilized)
• NADH Standard (Lyophilized)
• KGDH Positive Control
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

α-Ketoglutarate Dehydrogenase (α-KGDH) (EC 1.2.4.2) is a key enzyme in the citric acid cycle. It forms an enzyme complex with dihydrolipoamide succinyl transferase (E2) and dihydrolipoamide dehydrogenase (E3). α-KGDH converts α-ketoglutarate into succinyl-CoA in the presence of NAD and CoA. It is highly regulated by intracellular ATP/ADP and NADH/NAD ratios and calcium. In humans, decreased KGDH activity can lead to neurodegenerative diseases such as Alzheimer’s disease. Recent studies show that α-KGDH is a target of oxidative stress; reactive oxygen species (ROS) inhibit KGDH activity which diminishes its critical function and can cause a bioenergetic deficit. BioVision’s α-KGDH assay kit provides a quick and easy way for monitoring α-KGDH activity in various samples. In the assay, α-KGDH converts α-ketoglutarate into an intermediate which reduces the probe to a colored product with strong absorbance at 450 nm. The assay is simple, sensitive and can detect α-ketoglutarate dehydrogenase activity lower than 0.1 mU in a variety of samples.


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Is it better to use mitochondria or cell lysate? Cell lsyate is showing lot of background.
1. Use mitochondria instead of whole cell lysate. There can be a lot of NADH in the cell lysate that creates background. Mitochondria will help enrich the sample with mor active alpha KDGH per ul of sample.2. You canfilter the cell lysate samples through a 10KDa mol. wt cut off filter column. NADH, being a small molecule will flow through and the retentate within the filter insert will have the high mol. wt. proteins/enzymes including KDGH.
Can frozen samples be used with this assay?
Fresh samples are always preferred over frozen samples. 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.
Are trial sizes of this kit available?
Unfortunately, we do not have trial sizes of this kit available. However,for customers based in the US or Canada, a 10% off list price introductory discount can be offered on the list price. For all other customers based out of this area our regional BioVision distributor should be contacted.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Can alternate buffers be used for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
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.
Chaumeil et al., Hyperpolarized [1-13C] Glutamate: A Metabolic Imaging Biomarker of IDH1 Mutational Status in Glioma. Cancer Res., Aug 2014; 74: 4247 - 4257.
For more citations of this product click here