Citrate Assay Kit

(Catalog #K655-100; 100 assays; Store Kit at -20°C)

I. Introduction:

Citric acid (HOOC-CH₂-C(-OH)(-COOH)-CH₂-COOH) is a key intermediate in the TCA cycle which occurs in mitochondria. It is formed by the addition of oxaloacetate to the acetyl group of acetyl-CoA derived from the glycolytic pathway. Citrate can be transported out of mitochondria and converted back to acetyl CoA for fatty acid synthesis. Citrate is an allosteric modulator of both fatty acid synthesis (acetyl-CoA carboxylase) and glycolysis (phospho- fructokinase). Citrate is widely used industrially in foods, beverages and pharmaceuticals. Citrate metabolism and disposition can vary widely due to sex, age and a variety of other factors. BioVision's Citrate Assay Kit provides a simple, sensitive and rapid means of quantifying citrate in a variety of samples. In the assay, citrate is converted to pyruvate via oxaloacetate. The pyruvate is quantified by converting a nearly colorless probe to an intensely colored (λ_max=570 nm) and fluorescent (E_x/E_m, 535/587 nm) product. The Citrate Assay Kit can detect 0.1 to 10 nmoles (~2 µM-10 mM) of citrate in a variety of samples.

Kit Contents:

1. 
   

   Storage and Handling:

   
   

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

2. 
   

   Reagent Preparation and Storage Conditions:

   
   

Citrate Probe: Reconstitute with 0.3 ml of DMSO (warm to >18°C to liquefy before use) and mix thoroughly. Protect from light and moisture. Store at -20°C. Use within 2 months. 

Citrate Developer, Enzyme Mix: Dissolve with 220 µl Assay Buffer separately. Pipette up and down to dissolve. Aliquot into portions, store at –20^oC. Avoid repeated freeze/thaw cycles. Use within 2 months.

Citrate Standard: Dissolve in 100 µl dH2O to generate 100 mM (100 nmol/µl) Citrate Standard solution. Keep on ice while in use. Store at -20°C.

3. 
   

   Assay Protocol:

   
   

1. Standard Curve Preparations:

Colorimetric Assay: Dilute the Citrate Standard to 1 nmol/l by adding 10 µl 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.

Fluorometric Assay: Dilute the Citrate standard to 0.1 nmol/µl by adding 10 µl of the standard to 990 µl of dH2O, mix well, then further dilute by adding 10 µl to 90 µl of dH2O. Add 0, 2, 4, 6, 8, 10 µl into a series of standards well on a 96-well plate. Adjust the volume to 50 µl/well to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well.

1. 
   

   Sample Preparation:

   
   

Tissue (20 mg) or cells (2 x 10⁶) should be rapidly homogenized with 100 µl of Citrate Assay Buffer. Centrifuge at 15,000 g for 10 minutes to remove cell debris. 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 µl 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 standard curve range.

2. 
   

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

   
   

Colorimetric Assay Fluorometric Assay

Sample Bkgd Control* Sample Bkgd Control*

Citrate Assay Buffer 44 µl 46 µl 44 µl 46 µl

Citrate Enzyme Mix 2 µl ---- 2 µl ----

Developer 2 µl 2 µl 2 µl 2 µl

Citrate Probe** 2 µl 2 µl 2 µl 2 µl 

*Samples may contain oxaloacetate or pyruvate which can generate a background and need to be subtracted from the sample background signal.

**For the fluorometric assay, dilute 10X with DMSO to reduce fluorescent background.

3. 
   

   Add 50 µl of the Reaction Mix to each well containing the Citrate Standard and test samples. Add 50 µl of the sample background control mix to background control wells.

   
   


4. 
   

   Incubate for 30 minutes at room temperature, protect from light.

   
   


5. 
   

   Measure OD at 570 nm or fluorescence at E_x/E_m 535/587nm.

   
   


6. 
   

   Calculation: Correct background by subtracting the value of the 0 Citrate Standard from all readings. Next subtract the value of the Sample Background Control from the test sample. Plot the standard curve. Apply corrected sample readings to the standard curve to get Citrate amount in the sample wells. The Citrate concentrations in the test samples:

   
   

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

Where: Ay is the amount of citrate (nmol) in your sample from the standard curve. Sv is the sample volume (l) added to the sample well.

Citric acid molecular weight: 191 g/mol.