L-Amino Acid Quantitation Kit

I. Introduction:

L-Amino acids are the most essential elements in biology. Accurately quantitating L-amino acids in body fluids or purified samples may provide valuable information for diagnostic or basic research studies. BioVision’s L-Amino Acid Assay Kit provides a convenient means for directly detecting L-amino acid in biological samples. There is no requirement for sample pretreatment or purification when using this kit. The L-amino acid(s) level can be quantified using fluorometric (at Ex/Em = 535/587 nm) or colorimetric (at l = 570 nm) methods in 96-well plates.  

1. 
   

   Kit Contents:

   
   

*Mixture of all amino acids at equal molar ratio. Glycine can’t be detected by this kit.

2. 
   

   Reagent Preparation and Storage Conditions:

   
   

Probe: Dissolve the probe with 220 μl of dried DMSO (provided) before use. Mix, store at –20C, protect from light and moisture. Use within two months.

Enzyme Mix: Dissolve in 220 μl L-Amino Acid Assay Buffer. Pipette up and down to complete dissolve the content. Store at –20C. Use within two months.

3. 
   

   Assay Protocol:

   
   

1. Standard Curve Preparations: For colorimetric assay, add 0, 2, 4, 6, 8, 10 μl L-Amino Acid Standard into each well individually of a 96-well plate to generate 0, 8, 16, 24, 32, 40 nmol/well of L-Amino Acid Standard. Adjust volume to 50 μl/well with L-Amino Acid Assay Buffer.

For fluorometric assay, dilute the L-Amino Acid to 0.4 nmol/μl by adding 10 μl of the L-Amino Acid to 90 μl of L-Amino Acid Assay Buffer, mix well. Add 0, 2, 4, 6, 8, 10 μl into each well individually to generate 0, 0.8, 1.6, 2.4, 3.2, 4.0 nmol/well of the L-Amino Acid Standard. Adjust volume to 50 μl/well with L-Amino Acid Assay Buffer.

1. 
   

   Sample Preparations: Prepare test samples in 50 μl/well with L-Amino Acid Assay Buffer in the 96-well plate. We suggest using several doses of your sample to ensure the readings are within the standard curve range.

   
   

3. Reaction Mix Preparation: Mix enough reagents for the number of assays performed: For each well, prepare a total 50 μl Reaction Mix containing:

46 μl L-Amino Acid Assay Buffer

2 μl L-Amino Acid Probe

2 μl L-Amino Acid Enzyme Mix,

Mix well.

2. 
   

   Add 50 μl of the Reaction Mix to each well containing the L-Amino Acid standard or test samples.

   
   


3. 
   

   Incubate the reaction for 30 minutes at 37^oC, protect from light.

   
   


4. 
   

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

   
   


5. 
   

   Correct background by subtracting the value of the 0 L-amino acid control from all samples (The background reading can be significant and must be subtracted from sample readings). Then apply the sample readings to the L- amino acid standard curve to obtain the total amino acid amount.

   
   

L-Amino Acid Concentration = A/Sv (nmol/μl or mM)

Where A: L-Aamino acid amount (nmol) from the standard curve based on Absorbance OD570 or fluorescence of your samples.

Sv: Sample volume (μl) you added into the sample wells.

L-Amino Acid Standard Curve. Different doses of L-Amino Acids were measured according to the kit procedure.