Alanine Assay Kit

1. 
   

   Introduction:

   
   

Alanine is the 2^nd most abundant of the 20 proteinogenic amino acids. Nonessential, being available from dietary sources, it plays a key role in the glucose-alanine cycle between tissues and liver. In muscle and other tissues that degrade amino acids, amino groups are pooled as glutamate by transamination. Glutamate then transfers the amino group to pyruvate via alanine aminotransferase, forming alanine and α-ketoglutarate. The alanine is passed into the blood and transported to the liver. A reverse of the alanine aminotransferase reaction takes place in liver. Pyruvate can be used in gluconeogenesis, to form glucose which may return to other tissues through the circulatory system. There appears to be a correlation between alanine levels and and higher blood pressure, energy intake, cholesterol levels, and body mass index. BioVision’s Alanine Assay Kit provides a sensitive detection method of alanine. In the kit, alanine is converted to pyruvate which is specifically detected leading to proportional color (λ=570nm: 0-10 nmol) or fluorescence (Ex/Em 535/587nm: 0-1 nmol) development. Serum concentration: ~24-76 µg/ml (~3-9 nmol/10 µl).

2. 
   

   Kit Contents:

   
   

Components	K652-100	Cap Code	Part No.
Alanine Assay Buffer Alanine probe DMSO Alanine Converting Enzyme Alanine Development Mix Alanine Standard (10 mol)	25 ml lyophilized 0.4 ml lyophilized lyophilized lyophilized	WM Red Brown Purple Green Yellow	K652-100-1 K652-100-2 K652-100-3 K652-100-4 K652-100-5 K652-100-6

3. 
   

   Storage and Handling:

   
   

Store the kit at -20^oC, protect from light. Allow Assay Buffer to warm to room temperature before use. Briefly centrifuge vials before opening. Read the entire protocol before performing the assay.

4. 
   

   Reagent Reconstitution and General Consideration:

   
   

Alanine Probe: Add 220 µl DMSO. Pipette up and down to dissolve. Protect from light and moisture. Stable for 2 months at -20°C.

Alanine Converting Enzyme, Development Enzyme Mix: Dissolve separately with 220 l dH₂O. Pipette up and down to dissolve. Aliquot into portions and store at –20^oC. Avoid repeated freeze/thaw cycles. Use within two months.

Alanine Standard: Dissolve in 100 l dH2O to generate 100 mM (100 nmol/l) Alanine Standard solution. Keep cold while in use. Store at -20°C.

Ensure that the Assay Buffer is at room temperature before use. Keep the Alanine Enzyme Mix on ice during the assay and protect from light.

5. 
   

   Alanine Assay Protocol:

   
   

1. 
   

   Alanine Standard Curve:

   
   

Colorimetric: Dilute 10 l of the 100mM Alanine standard with 990 l DI H₂O to generate 1 mM standard Alanine. Add 0, 2, 4, 6, 8, 10 l of the diluted Alanine standard into a 96-well plate to generate 0, 2, 4, 6, 8, 10 nmol/well standard. Bring the volume to 50 l with Assay buffer.

Fluorimetric: Dilute standard as for the colorimetric procedure, then take 100 µl of the 1 mM standard and add to 900 µl DI H₂O to make 0.1mM Alanine standard. Add 0, 2, 4, 6, 8, 10 l of the diluted Alanine standard into a 96-well plate to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well standard. Bring the volume to 50 l with Assay buffer.

 

 

 

 

 

 

2. 
   

   Sample Preparation:

   
   

 

Tissues or cells (1×10⁶) can be homogenized in 100 l Assay Buffer centrifuge to remove insoluble material at 13,000 g, 10 minutes. 10-50 l deproteinized serum samples can be directly diluted in the Assay Buffer. Bring sample wells to 50 μl/well with Assay Buffer in a 96-well plate. For unknown samples, we suggest testing several doses of your sample to make sure the readings are within the standard curve range.

3. 
   

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

   
   

Alanine Measurement Background Control*

44 μl Assay Buffer 46 µl Assay Buffer

2 μl Alanine Converting Enzyme ----------------------

2 μl Alanine Development Mix 2 μl Alanine Development Mix

2 μl Alanine Probe** 2 μl Alanine Probe

* Use background control if high levels of pyruvate are suspected to be in the samples.

** For the fluorescent assay dilute the probe 10X to reduce background. 

Add 50 μl of the Reaction Mix to each well containing the Alanine Standard and test samples. Mix well. Incubate the reaction for 30 min at 37°C, protect from light.

4. 
   

   Measure O.D. at 570 nm in a microplate reader or fluorescence using Ex/Em 535/587 nm.

   
   


5. 
   

   Calculation: Correct background by subtracting the value derived from the 0 Alanine control from all sample readings (The background reading can be significant and must be subtracted from sample readings). Plot Alanine standard Curve, Alanine concentrations of the test samples can then be calculated:

   
   

C = S_a/S_v nmol/μl, or mM

where S_a is the sample amount of unknown (in nmol) from standard curve,

S_v is sample volume (μl) added into the wells.

L-Alanine Molecular Weight is 89.1 g/mol.