Alpha-Ketoglutarate Assay Kit

(Catalog #K677-100; 100 assays; Store Kit at -20°C)

 

I. Introduction:

α-Ketoglutarate (α-KG) is a key intermediate in the Krebs cycle, coming after isocitrate and before succinyl CoA. Anaplerotic reactions replenish the cycle by synthesizing α-KG from transamination of glutamate, or through the action of glutamate dehydrogenase. α-KG is an important nitrogen transporter. Being a key intermediate, it is one of the organic acids measured in newborns as an indicator of inborn errors of metabolism. BioVision's α-Ketoglutarate Assay Kit provides a simple, sensitive and rapid means for quantifying α-KG in a variety of samples. In the assay, α-ketoglutarate is transaminated with the generation of pyruvate which is utilized to convert a nearly colorless probe to both color (λ_max = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The α-Ketoglutarate Assay Kit is useful for detecting α-ketoglutarate in the range of 0.01 to 10 nmoles.

II. Kit Contents:

Components	K677-100	Cap Code	Part Number
α-KG Assay Buffer α-KG OxiRed Probe DMSO (Anhydrous) α-KG Converting Enzyme α-KG Development Enzyme Mix α-KG Standard (10 mol)	25 ml lyophilized 0.4 ml lyophilized lyophilized lyophilized	WM Red Brown Purple Green Yellow	K677-100-1 K677-100-2 K677-100-3 K677-100-4 K677-100-5 K677-100-6

1. 
   

   Storage and Handling:

   
   

Store kit at –20^oC, protect from light. Warm α-KG Assay Buffer to room temperature before use. Briefly centrifuge all small vials prior to opening.

2. 
   

   Reagent Preparation and Storage Conditions:

   
   

α-KG Probe: Add 220 µl DMSO. Pipette up and down to dissolve. Protect from light and moisture. Stable for 2 months at -20°C.

α-KG Converting Enzyme, α-KG Enzyme Mix: Dissolve with 220 l α-KG Buffer separately. Pipette up and down to dissolve. Aliquot into vials with sufficient amount for each experiment and store at –20^oC. Avoid repeated freeze/thaw cycles. Use within two months.

α-KG Standard: Dissolve in 100 l dH2O to generate 100 mM (100 nmol/l) α-KG Standard solution. Keep cold while in use. Store at -20°C.

3. 
   

   Assay Protocol:

   
   

1. Standard Curve Preparations:

Dilute the α-KG Standard to 1 nmol/l by adding 10 l of the Standard to 990 l of dH2O, mix well. Add 0, 2, 4, 6, 8, 10 l into a series of standards wells on a 96 well plate. Adjust volume to 50 l/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of the Standard.

1. 
   

   Sample Preparation:

   
   

Tissue (20 mg) or cells (2x10⁶) are rapidly homogenized with 100 l of ice cold PBS or other buffer (pH 6.5-8). Enzymes in samples may interfere with the assay. We suggest deproteinizing samples using a perchloric acid/KOH protocol (BioVision, Cat.# K808-200) or 10 kD molecular weight cut off spin columns (BioVision, Cat # 1997-25). Add 1-50 l samples into duplicate wells of a 96-well plate and bring volume to 50 µl with Assay Buffer. We suggest testing several doses of your samples to ensure readings are within the linear range.

 

 

 

 

2. 
   

   Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 µl Reaction Mix containing:

   
   

Sample Bkgd. Control*

α-KG Assay Buffer 44 µl 46 µl

α-KG Converting Enzyme 2 µl -----

α-KG Enzyme Mix 2 µl 2 µl

α-KG probe 2 µl 2 µl

 

Add 50 µl of the Reaction Mix to each well containing the α-KG Standard, samples or background control*.

*Note: Pyruvate generates background. If pyruvate is suspected in your sample, you can do the background control omitting the converting enzyme. The background control can be subtracted from the α-KG reading.

3. 
   

   Incubate for 30 minutes at 37°C, protect from light.

   
   


4. 
   

   Measure OD at 570 nm or fluorescence using Ex/Em=535/587 nm.

   
   


5. 
   

   Calculation: Correct background by subtracting the value of the 0 α-KG blank from all readings. Plot the standard curve. Apply the corrected sample readings to the standard curve to get α-KG amount in the sample wells. The α-KG concentrations in the test samples:

   
   

C = Ay/Sv (nmol/l; or mol/ml; or mM)

Where: Ay is the amount of α-KG (nmol) in your sample from the standard curve.

Sv is the sample volume (l) added to the sample well.

α-Ketoglutarate molecular weight: 146.11