Acid Phosphatase Colorimetric Assay Kit

(Catalog# K411-500; 500 reactions; Store kit at –20^oC)

1. 
   

   Introduction:

   
   

Acid phosphatases (AP) dephosphorylate phosphate groups from phosphate esters under acid conditions. Different acid phosphatase isozymes are found in different organs, and their serum levels are used as a diagnostic for disease in the corresponding organs. 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 Assay Kit is a high sensitivity, simple, direct and HTS-ready colorimetric assay designed to measure AP activity in serum and other samples. It is suitable for research and drug discovery. The kit uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate which turns yellow (λ_max = 405 nm) when dephosphorylated by AP. The kit can detect µU acid phosphatase activity in samples.

Components	K411-500	Cap Code	Part No.
AP Assay Buffer pNPP Substrate (10 TAB) AP Enzyme Stop Solution	100 ml 1 vial 1 vial 10 ml	NM Red Green WM	K411-500-1 K411-500-2 K411-500-3 K411-500-4

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 before performing the assay.

4. 
   

   Reagent Reconstitution and General Consideration:

   
   

pNPP Solution: Dissolve 2 tablets pNPP into 5.4 ml Assay Buffer to generate 5 mM pNPP. Two tablets are sufficient for 100 assays. NEVER TOUCH THE TABLETS WITH BARE HANDS. The pNPP solution is stable for 12 hours on ice.

AP Enzyme Solution: Reconstitute AP Enzyme with 1 ml Assay Buffer. NEVER FREEZE! The enzymes are stable for up to 2 months at 4^oC after reconstitution.

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

5. 
   

   Acid Phosphatase Assay Protocol:

   
   

0. 
   

   Sample Preparations:

   
   

Inhibitors of AP, such as 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, 3 minutes. Add test samples directly into 96-well plate, bring total volume to 80 l with Assay Buffer.

If samples contain color, it may interfere with O.D. 405 nm readings, use a sample background control. Add the same amount of sample into separate wells, bring volume to 80 µl. Add 20 l stop solution and mix well to terminate AP activity in the sample.

 

 

 

 

 

1. 
   

   Add 50 μl of pNPP Substrate Solution to each well containing the test samples and background controls. Mix well. Incubate the reaction for 60 min at 25°C, protect from light.

   
   


2. 
   

   Standard Curve:

   
   

Dilute 40 l of the 5 mM pNPP solution with 160l Assay Buffer to generate 1 mM pNPP standard. Add 0, 4, 8, 12, 16, 20 l into 96-well plate in duplicate to generate 0, 4, 8, 12, 16, 20 nmol/well pNPP standard. Bring the final volume to 120 l with Assay Buffer.

Add 10 μl of AP enzyme solution to each well containing the pNPP standard. Mix well. The AP enzyme will convert pNPP substrate to an equal amount of colored p-Nitrophenol (pNP). Incubate the reaction for 60 min at 25°C, protect from light.

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 the sample was prepared in step 1), gently shake the plate. Measure O.D. 405 nm in a micro-plate reader.

   
   


4. 
   

   Calculation: Correct background by subtracting the value derived from the 0 standard from all the standards, samples and sample background controls (The background reading can be significant and must be subtracted from sample readings). Plot pNP standard Curve. Apply sample readings to the standard curve to get the amount of pNP generated by AP sample. AP activity of the test samples can then be calculated:

   
   

AP activity (U/ml) = A/V/T

Where A is amount of pNP generated by samples (in mol).

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

T is reaction time (in minutes)

Unit Definition: One unit of AP is the amount of enzyme causing the hydrolysis of one micromole of pNPP to pNP per minute at pH 5.0 and 25°C.