Urea Assay Kit

• Post Date:

  Sep 10,2015

• Expiry Date:

  Sep 09,2016

• Detailed Description:

  Cas No. :RNF91006 Specs:95%
  Payment Method: tt
  Product Description
  REAGEN? Urea Assay Kit is a cuvette-based enzymatic assay for the determination of urea across a variety of food samples. The kit uses a spectrophotometric, kinetic assay to detect the enzymatic degradation of urea directly from food samples.
  The unique features of the kit are:
  
  ? High sensitivity and low detection limit (0.6 ppm)
  ? High reproducibility
  ? A rapid (30 minute), robust enzyme-based assay
  
  Procedure Overview
  REAGEN? Urea Assay Kit uses a coupled enzymatic reaction scheme to detect urea in food samples. The assay measures the concentration of urea using the urease enzyme which converts urea to ammonia. The ammonia produced by this reaction is then used as a substrate by the enzyme glutamate dehydrogenase to make glutamate. In the process, NADH is stoichiometrically converted to NAD+. This conversion, measured at 340 nm, is proportional to the level of urea in the sample.
  
  The absorbance of each sample at 340 nm is measured using a spectrophotometer. The concentration of urea in each sample is then directly determined from the change in NADH absorbance at 340 nm within 25 minutes.
  
  Kit Contents, Storage and Shelf Life
  REAGEN? Urea Assay Kit has the capacity for 48 determinations or 24 determinations in duplicate. Store the kit at 4°C. The shelf life of the kit is 12 months, after receipt, when the kit is properly stored.
  Kit Contents Amount Storage
  Substrate Solution One vial 4oC
  Urease 0.25 mL 4oC
  Microtiter plate One Room temp or 4oC
  
  
  Sensitivity (Detection Limit)
  
  Sample Volume Detection Limit (mg/L)
  0.01 mL 12
  0.2 mL 0.6
  The linear range of the assay is 0.6 – 600 mg/L (ppm).
  
  Required Materials Not Provided With the Kit
  
  ? Spectrophotometer (340 nm)
  ? Low volume (0.3 mL) UV cuvettes, such as Brand Scientific catalog #7592 00
  ? Deionized or distilled water
  ? 50 mL plastic conical tubes
  
  Warnings and Precautions
  
  ? Do not use the kit past the expiration date.
  ? Try to maintain a laboratory temperature of 20°–25°C (68°–77°F). Avoid running assays under or near air vents, as this may cause excessive cooling, heating and/or evaporation. Also, do not run assays in direct sunlight, as this may cause excessive heat and evaporation. Cold bench tops should be avoided by placing several layers of paper towel or some other insulate material under the assay plates during incubation.
  ? Make sure you are using only distilled or deionized water since water quality is very important.
  ? When pipetting samples or reagents into an empty microtiter plate, place the pipette tips in the lower corner of the well, making contact with the plastic.
  
  
  SAMPLE PREPARATION
  General information on sample preparation
  ? Grind or homogenize solid samples then extract with water and filter to clarify samplexer.
  ? Adjust acidic samples to pH 7.5 using NaOH solution. Allow pH of carbonated samples to stabilize for 15 minutes before final pH adjustment.
  
  ? Measure colored samples against a sample blank containing only buffer plus sample (using the same sample volume used for the assay). Strongly colored samples can be treated with polyvinylpolypyrrolodine (PVPP, final concentration about 1%) to decolorize: Stir 1 minute and filter to remove solids.
  ? Extract samples containing fat with hot water. Cool the extract and remove the aqueous sample from the fat layer.
  ? Protein rich samples such as milk-based samples should be deproteinized before performing the assay. Use a Carrez based deproteinization since, unlike acid-based deproteinization procedures, it will not hydrolyze polysaccharides in the sample
  Carrez deproteinization: Transfer 0.5 – 5 g sample to a 50 mL volumetric flask containing about 30 mL dH2O. Add 2.5 mL Carrez I solution (3.6% potassium hexacyanoferrate (II) ? 3 H2O) and 2.5 mL Carrez II solution (7.2% zinc sulfate ? 7 H2O). Mix and carefully adjust the pH to 8 with sodium hydroxide, adjust the volume to 50 mL, mix and filter.
  Beer
  To remove the carbonation (bubbles), stir 10 mL of beer in a beaker for 30 – 40 seconds with a glass rod or filter using a fluted filter paper. The “decarbonated” sample can be used undiluted in the assay
  Fruit Juices
  Juice samples can be treated with polyvinylpolypyrrolodine (PVPP, final concentration about 5%) to decolorize: Stir 1 minute and filter to remove solid
  Fruit Jam
  Accurately weigh about 0.5 g sample and transfer to a 100 mL volumetric flask containing about 99 mL dH2O. Mix, adjust to 100 mL volume and filter
  Condensed Milk
  Accurately weigh about 0.5 g sample and transfer to a 50 mL volumetric flask containing about 30 mL dH2O. Mix and incubate 10 minutes at room temperature. Add 2.5 mL Carrez I solution (3.6% potassium hexacyanoferrate (II) ? 3 H2O) and 2.5 mL Carrez II solution (7.2% zinc sulfate ? 7 H2O). Mix and carefully adjust the pH to 8 with sodium hydroxide, adjust the volume to 50 mL, mix and filter to clarif
  
  
  Ice Cream
  Accurately weigh about 0.5 g sample and transfer to a 100 mL volumetric flask c ontaining about 99 mL dH2O. Mix, adjust to 100 mL volume and filte
  Potato-based Foods, Fruits and Vegetables
  Homogenize > 25 g sample (using a blender or other homogenizer). Transfer 10 g to a 50 mL tube and add 30 mL dH2O. Vortex mix or shake for 3 minutes. Add 1.25 mL Carrez I solution (3.6% potassium hexacyanoferrate (II) ? 3 H2O) and 1.25 mL Carrez II solution (7.2% zinc sulfate ? 7 H2O). Mix and carefully adjust the pH to 7.5 with sodium hydroxide, add 50 μL n-butanol and adjust the volume to volume to 50 mL
  
  
  UREA DETECTION PROTOCOL
  
  Reagent Preparation
  
  IMPORTANT: All reagents should be brought up to room temperature before use (1 – 2 hours at20 – 25°C / 68 – 77°F); Make sure you read “Warnings and Precautions” section on page 3.
  
  Reconstitute the Substrate Solution by adding 10 mL dH2O to the vial. Gently swirl or invert the vial ten times to dissolve the powder.
  
  ELISA Testing Protocol
  
  Note: Each determination is performed in 0.3 mL microcuvettes, in parallel, using samples diluted into water without urease enzyme. The color change of these no-enzyme controls (Sample blank) is then subtracted from the color change of corresponding enzyme reactions (Sample reaction) to correct for sample background signals.
  
  
  Pipet into low volume cuvettes
  Sample
  blank Sample
  reaction
  Sample
  dH20
  
  Note: Sample volume + dH20 volume should equal 200 μL.
  
  Substrate Solution
  10 μL
  190 μL
  
  
  
  95 μL 10 μL
  190 μL
  
  
  
  95 μL
  Mix and incubate 5 min at 37°C or room temperature. Measure the absorbance at 340 nm (A1).
  
  Urease
  dH2O ----
  5 μL 5 μL
  ----
  Mix and incubate 10 min at 37°C or 25 min at room temperature. Measure the absorbance at 340 nm (A2).
  
  Determination of Urea in Food Samples
  To calculate the urea concentration, use the following general equation:
  
  c = V x MW x ΔA (g / L)
  ε x d x v
  
  V = final volume (0.3 mL)
  v = sample volume (0.01 – 0.2 mL)
  MW = molecular weight of urea (60.06g/mol)
  d = light path (1 cm)
  ε = extinction coefficient of NADH (6220 at 340 nm)
  
  ΔA = (A1 – A2)sample – (A1 – A2)blank
  
  For example, for a 10 μL sample:
  
  c = 0.3 x 60.06 x ΔA (g / L) = 0.289 x ΔA (g urea / L sample solution)
  6220 x 1 x 0.01
  
  If the sample has been diluted during the sample preparation protocol, the result must be multiplied by the dilution factor (F).
  
  Alterative Protocol using a 96-well microplate
  
  Note: The microplate protocol uses a reaction volume of 300 μL.
  
  1. Add 10 μL to 200 μL of sample solution, depending on the sample type, in duplicate into microplate wells.
  2. Add dH2O into the wells for a total volume of 200 μL in each well.
  3. Add 95 μL of Substrate Solution into the wells. Immediately measure the absorbance of each sample at 340 nm (A1).
  4. Begin the reaction by adding 5 μL of Urease into one of the duplicate wells (Sample Reaction). Mix. Add 5 μL of dH2O into the other duplicate well (Sample Blank). Mix. Incubate the plate at room temperature; after 30 min, read the absorbance at 340 nm (A2).
  
  Data analysis is performed in the manner described in the previous section.
  
  

• CAS Registry Number:

  RNF91006

• Company:

  REAGEN LLC     [ United States ]        

• Contact:

  Nicole

• Tel:

  8568585434

• Fax:

  1-302-269-1897

• Email:

  reagenkits@gmail.com