Phenylalanine Assay Kit

I. Introduction:

L-Phenylalanine (PHE) is an electrically-neutral amino acid, one of the twenty common and one of the three aromatic amino acids used to biochemically form proteins. Phenylalanine uses the same active transport channel as tryptophan to cross the blood-brain barrier, and, in large quantities, interferes with the production of serotonin. Errors in PHE metabolism lead to phenylketonuria or PKU which can have dire consequences. BioVision’s Phenylalanine Assay Kit provides a quick, simple, accurate method for quantifying PHE in biological samples. In the assay, PHE is reductively deaminated with the simultaneous formation of NADH which reacts with our fluorescent probe to generate fluorescence at Ex/Em=535/587 nm. The assay is linear in the range from 0.1 to 1.0 nmol (2-20 M) of Phenylalanine.

II. Kit Contents:

1. 
   

   Storage and Handling:

   
   

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

2. 
   

   Reagent Preparation and Storage Conditions:

   
   

Tyrosinase, Enzyme Mix, Developer: Dissolve with 220 l Assay Buffer separately. Pipette gently to dissolve. Keep on ice. Store at –20^oC. Stable for at least two months

Phenylalanine Standard: Dissolve in100 µl dH₂O to generate a 10mM solution. Store at -20°C.

3. 
   

   Phenylalanine Assay Protocol:

   
   

0. 
   

   Standard Curve Preparations: Dilute the Phenylalanine Standard to 0.1 mM by adding 10 l of the Standard to 990 l of dH2O, mix well. Add 0, 2, 4, 6, 8, 10 l to a series of wells. Adjust volume to 50 l/well with Assay Buffer to generate 0, 0.2, 0.4, 0.6, 0.8 and 1 nmol per well of the Phenylalanine Standard.

   
   


1. 
   

   Sample Preparation: Tissue (20 mg) or cells (2x10⁶) can be homogenized in 100 l Assay Buffer, spin 15000g for 10 min to remove the insoluble material. Phenylalanine concentrations can vary over a rather wide range (normal range: 4-250µM to over 1mM in serum in pathological states. Serum should be deproteinized either using a deproteinizing kit (BioVision #K808-200) or a 10 kd molecular weight cut off spin filter (BioVision #1997-25). Take samples between 1-50 µl and adjust the well volume to 50 l with Assay Buffer. For unknown samples, it may be necessary to test several different doses to ensure the readings are within the range of the standard curve.

   
   


2. 
   

   Sample pretreatment:

   
   

The enzyme used in the assay can react with tyrosine and methionine as well as phenylalanine. Serum methionine concentrations are generally low enough to be insignificant in this assay. Tyrosine concentrations may interfere. If tyrosine interference is a

concern, add 5 µl tyrosinase to the samples and preincubate for 10 minutes before performing the assay to remove tyrosine interference.

4. 
   

   Development:

   
   

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

Assay Buffer 46 µl

Enzyme Mix * 2 µl

Developer 2 µl

Mix and add 50 µl of the Reaction Mix to each well containing Phenylalanine Standard or samples.

Note: * NADH, NADPH will generate background for the assay. In the absence of Enzyme Mix, the assay can detect NADH and NADPH background, replace the Enzyme Mix with 2 l of Assay Buffer. The background reading can be subtracted from Phenylalanine readings.

5. 
   

   Incubate at 37° for 20 minutes, protect from light.

   
   


6. 
   

   Measure fluorescence (Ex/Em 535/587 nm) in a plate reader.

   
   


7. 
   

   Calculation: Correct background by subtracting the 0 Phenylalanine standard from all readings (Note: The background can be significant and must be subtracted). Plot standard curve nmol/well vs. standard readings. Apply sample readings to the standard curve to get the amount of Phenylalanine in the sample wells. The Phenylalanine concentration in the test samples:

   
   

C = Ay/Sv (nmol/l; or mM)

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

Phenylalanine molecular weight: 165.2 g.mol^-1