Acid Phosphatase Fluorometric Assay Kit

(Catalog #K421-500; 500 reactions; Store kit at –20^oC)

1. 
   

   Introduction:

   
   

Acid phosphatase (AP) dephosphorylates phosphate groups from the phosphate esters in acid conditions. Different forms of acid phosphatase are found in different organs, and their serum levels are used as a diagnosis for disease in the corresponding organs. For example, elevated prostatic acid phosphatase levels may indicate the presence of prostate cancer and elevated tartrate resistant acid phosphatase levels may indicate the bone disease. BioVision’s Acid Phosphatase Fluorometric Assay Kit uses non-fluorescent Methylumbelliferyl phosphate disodium (MUP) as the substrate which has Ex/Em=360/440 nm when dephosphorylated by AP. The kit is an ultra-sensitive, simple, direct and HTS-ready fluorometric assay designed to measure AP activity in serum and other bio-samples. The detection sensitivity is ~1 U, more sensitive than colorimetric assays. The kit is suitable for both research and drug discovery.

Components	K421-500	Cap Code	Part No.
AP Assay Buffer MUP Substrate AP Enzyme Stop Solution	100 ml 1 vial 1 vial 25 ml	NM Red Green WM	K421-500-1 K421-500-2 K421-500-3 K421-500-4

2. 
   

   Kit Contents:

   
   


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 prior to performing the assay.

4. 
   

   Reagent Reconstitution and General Consideration:

   
   

MUP Solution: Dissolve MUP substrate into 1.2 ml Assay Buffer to generate 5 mM MUP substrate solution. The MUP solution is stable for 2 month at -20°C.

AP Enzyme: Reconstitute AP Enzyme with 1 ml Assay Buffer. The enzyme is stable for up to 2 months at 4°C after reconstitution. DO NOT FREEZE!

Ensure that the Assay Buffer is at room temperature before use. Keep samples and AP Enzyme on ice during the assay.

5. 
   

   Acid Phosphatase Assay Protocol:

   
   

0. 
   

   Sample Preparations:

   
   

Inhibitors of AP, like tartrate, fluoride, EDTA, oxalate, and citrate, should be avoided in sample preparation. Serum, plasma, urine, semen, and cell culture media can be assayed directly. Cells (1×10⁵) or tissue (~10 mg) can be homogenized in 100 l Assay Buffer, centrifuge to remove insoluble material at 13,000g for 3 minutes. Add test samples directly into 96-well plate, bring total volume to 110 l with Assay Buffer.

In order to avoid interference of other components in the sample, set a sample background control. Add the same amount of samples into separate wells, bring volume to 110 µl with Assay Buffer. Add 20 l Stop Solution and mix well to terminate AP activity in the sample.

 

1. 
   

   Dilute enough (1:10) 5 mM MUP substrate solution to 0.5 mM with Assay Buffer. Add 20 μl 0.5 mM MUP substrate solutions to each well containing the test samples and background controls. Mix well. Incubate the reaction for 30 min (or longer if AP activity in sample is low) at 25°C, protect from light.

   
   


2. 
   

   Standard Curve:

   
   

Dilute 10 l of the 5 mM MUP solution with 990 l Assay Buffer to generate 50 µM MUP standards. Add 0, 2, 4, 6, 8, 10 l into 96-well plate in duplicate to generate 0, 0.1, 0.2, 0.3, 0.4, 0.5 nmol/well MUP standard. Bring the final volume to 120 l with Assay Buffer.

Add 10 μl of AP enzyme solution to each well containing the MUP standard. Mix well. Incubate the reaction for 30 min at 25°C, protect from light. The ALP enzyme will convert MUP substrate to equal amount of fluorescent 4-Methylumbelliferone (4-MU).

3. 
   

   Stop all reactions by adding 20 μl Stop Solution into each standard and sample reaction except the sample background control reaction (since 20 l Stop Solution has been added into the background control when prepare the sample background control in step 1), gently shake the plate. Measure fluorescence intensity at Ex/Em 360/440 nm using a fluorescence microtiter plate reader.

   
   


4. 
   

   Calculation: Correct background by subtracting the value derived from the sample background controls. Plot 4-MU standard Curve. Apply sample readings to the standard curve to get the amount of 4-MU generated by AP sample. AP activity of the test samples can then be calculated:

   
   

AP Activity = A/V/T (mU/ml)

Where: A is amount of 4-MU generated by samples (in nmol).

V is volume of sample added in the assay well (in ml).

T is reaction time (in minutes).

Unit Definition: 1 unit of AP is the amount of enzyme causing hydrolysis of 1 mol of MUP per minute at 25^oC