Product Description:

alpha-Ketoglutarate (alpha-KG aka 2-oxoglutarate), the product of isocitrate dehydrogenase of the TCA (Krebs) cycle, represents a significant point of convergence in metabolism. Several amino acids can be converted to glutamate, which if transaminated or oxidatively deaminated, yields alpha-KG. Conversely, these amino acids can be synthesized from alpha-KG. Thus, intracellular levels of alpha- KG fluctuate according to nitrogen and carbon availability. As a Krebs cycle metabolite, it regulates anabolic and catabolic substrate availability as well as ATP production. In addition, it is an obligatory co-substrate for alpha-KG- and iron-dependent dioxygenases, which are a large group of phylogenetically conserved enzymes involved in hydroxylation reactions. As a substrate of the hydroxylases, alpha-KG exerts an impact on prolyl/aspartyl/lysyl hydroxylations, which in turn regulates the stability of the HIF-1 and collagen synthesis. Notably, alpha-KG binds and regulates G protein function because it is a ligand for the GPCR GPR99/GPR80. The metabolic and extra-metabolic functions of alpha-KG thus offer many possibilities for therapeutic interventions. The alpha-KG Assay is based on the difference in the UV absorption spectra between the oxidized and reduced forms of NADP+/NADPH at 340 nm. As low as ~10 uM alpha-KG may be detected by the assay kit (assay range 10 – 200 uM). The assay requires a 0.1 ml Quartz cuvette for measuring changes in O.D.340 nm. Alternatively, a UV-transparent 96-well plate can be used.

Kit Components:

alpha-KG Assay Solution: 10 ml (for 100 wells), store at -70ºC

GLDH (50x): 0.2 ml, store at -70ºC

NADPH (100x): 0.1 ml (brown vial), store at -70ºC (light sensitive)

Ammonium (500x): 0.1 ml, store at -70ºC

20 mM alpha-KG standard: 0.2 ml, store at -20ºC or -70ºC

PEG Solution: 5 ml store at 4°C (pipette with a cut tip)

MSDS:

Tris, ammonium chloride, PEG

Related Kits:

L-Lactate Assay, ATP Assay, NAD+ Assay, Glutamine Assay, Glutamate Assay, Pyruvate Assay