Ascorbic Acid Quantification Kit

(Catalog #K661-100; 100 assays; Store at -20^oC)

I. Introduction:

Ascorbic Acid (Vitamin C) plays an important role in many biological processes. It is a potent anti-oxidant, anti-inflammatory, anti-viral agent, and an immune stimulant and is present is a wide variety of foods and biological specimens. It is important to be able to monitor ascorbic acid content in these different samples. BioVision’s Ascorbic Acid Assay Kit provides a rapid, simple, and sensitive means of detecting ascorbic acid in various biological samples. In this assay, our proprietary catalyst oxidizes ascorbic acid to produce a product that interacts with the ascorbic acid probe, generating color and fluorescence. Ascorbic acid can be easily determined by either colorimetric (spectrophotometry at  = 570 nm) or fluorometric (Ex/Em = 535/587 nm) methods. The assay can detect 0.01-10 nmol of ascorbic acid per assay in various samples.

1. 
   

   Kit Contents:

   
   

Components	K661-100	Cap Code	Part Number
Ascorbic Acid Assay Buffer Ascorbic Acid Probe (lyophilized) Dimethylsulfoxide (DMSO, Anhydrous) Catalyst Ascorbic Acid Enzyme Mix (lyophilized) Ascorbic Acid Standard (20 mole)	25 ml 1 vial 0.4 ml 0.5 ml 1 vial 1vial	WM Red Brown Blue Green Yellow	K661-100-1 K661-100-2 K661-100-3 K661-100-4 K661-100-5 K661-100-6

 

 

 

 

 

2. 
   

   Storage and Handling:

   
   

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

3. 
   

   Reagent Preparation:

   
   

Ascorbic Probe: Dissolve in 220 l anhydrous DMSO (provided) before use. Store at –20^oC, protect from light and moisture. Use within two months.

Ascorbic Acid Enzyme Mix: Dissolve in 220 l Ascorbic Acid Assay Buffer. Aliquot and store at –20^oC. Use within two months.

Ascorbic Standard: Dissolve in 200 l of distilled water to generate 100 mM Ascorbic Standard stock solution. Store at –20^oC. Use within two months.

Catalyst: Ready to use as supplied

4. 
   

   Ascorbic Acid Assay Protocol:

   
   

1. Standard Curve Preparations:

For the colorimetric assay, dilute the standard to 1 mM by adding 10 l of the 100 mM Ascorbic Acid Standard to 990 l of distilled water, mix well. Add 0, 2, 4, 6, 8, 10 l into each well individually. Adjust volume to 120 l/well with Ascorbic Acid Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of Ascorbic Acid Standard.

For the fluorometric assay, dilute the Ascorbic Acid Standard to 0.01- 0.1 mM with the Ascorbic Acid Assay Buffer (Note: Detection sensitivity is 10 to 100 fold higher for a fluorometric than a colorimetric assay). Follow the procedure for the colorimetric assay.

Note: Diluted ascorbic acid standard is unstable, use fresh dilution each time.

 

 

 

 

1. 
   

   Sample Preparation: Prepare test samples to a final volume of 120 l/well with Ascorbic Acid Assay Buffer in a 96-well plate. We suggest testing several doses of your sample to make sure the readings are within the standard curve range.

   
   

NOTE:

1) Due to high protein contents and other compounds present in serum we recommend using FRASC Ascorbic Acid Kit (K671-100) for serum samples.

2) Ascorbate is easily oxidized during sample preparation and great care must be exercised to achieve quantitative recovery.

2. 
   

   Catalyst: Add 100 l of catalyst to 900 l of distilled water and vortex well.

   
   


3. 
   

   Add 30 l of catalyst to each standard and sample well.

   
   


4. 
   

   Ascorbic Acid Reaction Mix: Mix enough reagents for the number of samples and standards to be performed: For each well, prepare a total 50 l Reaction Mix containing:

   
   

46 l Ascorbic Acid Assay Buffer

2 l Ascorbic Acid Probe

2 l Ascorbic Acid Enzyme Mix

5. 
   

   Mix well. Add 50 l of the Reaction Mix to each well containing the Ascorbic Acid Standard and test samples. Mix well.

   
   


6. 
   

   Protect from light, Color is developed within 3 minutes and stable for an hour.

   
   


7. 
   

   Measure O.D. 570nm for colorimetric assay or Ex/Em = 535/590 nm for fluorometric assay in a micro-plate reader.

   
   


8. 
   

   Correct background by subtracting the value derived from the 0 ascorbic acid standard from all sample readings (Note: The background reading can be significant and must be subtracted from sample readings). Apply sample readings to the generated standard curve. Ascorbic Acid concentration can then be calculated:

   
   

C = As / Sv nmol/l or mol/ml or mM

Where: As is ascorbic acid amount from standard curve (nmol).

Sv is the sample volume added in sample wells (l).

Ascorbic Acid molecular weight: 176.12.