Phosphate Fluorometric Assay Kit

(Catalog #K420-100; 100 Assays; Store kit at -20^oC)

1. 
   

   Introduction:

   
   

Inorganic phosphate (Pi) is one of the most important ions in biological systems. It functions in a variety of roles. One of the most important roles is as a molecular switch, turning enzyme activity on and off through the mediation of the various protein kinases and phosphatases in biological systems. A highly sensitive assay is desired to monitor Pi in variety samples or monitor Pi changes during kinase and phosphatase reactions. The newly designed Phosphate Assay Kit provides a sensitive, easy, quick means of assessing phosphate over a wide range of concentrations. In the assay, inorganic phosphate reacted with maltose to produce glucose in the present of a special enzyme. The glucose is specifically oxidized to generate a product that reacts with the OxiRed^® probe to generate fluorescence (Ex/Em=535/587nm). The kit can be used to detect Pi produced through reactions involving ATPases, GTPases, 5’-nucleotidase, protein phosphatases, acid and alkaline phosphatases, and phosphorylase, etc. from a variety of samples. Glucose background can be eliminated by doing a glucose control. Phosphate concentrations between 0.4 μM and 10 μM, with a lower detection limit of approximately 40 pmol can be directly determined.

Components	100 assays	Cap Color	Part Number
Phosphate Assay Buffer OxiRed® Probe Dimethylsulfoxide (DMSO; Anhydrous) Maltose Converter Glucose Developer Phosphate Substrate Phosphate Standard (100 mM)	25 ml 1 vial 0.4 ml 1 vial 1 vial 0.2 ml 50 µl	WM Red Brown Blue Green Purple Yellow	K420-100-1 K420-100-2 K420-100-3 K420-100-4 K420-100-5 K420-100-6 K420-100-7

2. 
   

   Kit Contents:

   
   


3. 
   

   Storage and Handling:

   
   

Store kit at –20°C, protect from light. Allow reagents to warm to room temperature and briefly centrifuge vials prior to opening. Read the entire protocol before the assay.

4. 
   

   Reagent Preparation:

   
   

OxiRed^® Probe: Dissolve in 220 μl DMSO (provided) before use. Store at –20°C, protect from light and moisture. Use within two months.

Maltose Converter, Glucose Developer: Dissolve in 220 μl Assay Buffer separately. Aliquot and store at –20°C. Use within two months.

5. 
   

   Phosphate Assay Protocol:

   
   

*Caution: Phosphate contamination in samples and buffers must be carefully avoided. Laboratory detergents can contain high concentrations of phosphates and glassware must be thoroughly rinsed with distilled water to remove any phosphate bound to the glass.

 

1. 
   

   Standard Curve Preparations:

   
   

Dilute the Phosphate Standard to 25M by adding 10 μl of the Phosphate Standard to 990 μl of Assay Buffer, mix well, then take 10 μl into 390 μl of Assay Buffer. Mix well. Add 0, 2, 4,

6, 8, 10 μl into each well individually. Adjust volume to 50 μl/well with Assay Buffer to generate 0, 50, 100, 150, 200, 250 pmol/well of the Phosphate Standard.

 

 

 

 

2. 
   

   Sample Preparations: Add 1 – 50 l test samples in a 96-well plate, bring the volume to total 50 μl/well with Assay Buffer. If using serum sample, serum*(0.5-2 μl/well) can be directly diluted in the Assay Buffer. We suggest testing several doses of your sample to make sure the readings are within the standard curve range.

   
   

*Serum contain 4-6 mM glucose, it will interfere with the result, please set a glucose control.

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:

   
   

45 μl Assay Buffer

0.2 μl OxiRed^® Probe

1 μl Phosphate Substrate

2 μl Maltose Converter*

2 l Glucose Developer.

*Note: Glucose interferes with Pi the reaction. If significant amount of glucose is in your sample, you may do a glucose control by omitting the Maltose Converter in the reaction, which will read glucose background only. The glucose background should be subtracted from Pi readings.

4. 
   

   Add 50 μl of the Reaction Mix to each well containing the Phosphate Standard and test samples, mix well.

   
   


5. 
   

   Incubate the reaction for 2 hours at 37°C, protect from light.

   
   


6. 
   

   Measure fluorescence at Ex/Em = 535/587 nm in a micro plate reader.

   
   


7. 
   

   Correct background by subtracting the value derived from the 0 phosphate control from all sample readings (Note: The background reading can be significant and must be subtracted from sample readings). Plot the Pi Standard Curve, apply the sample readings to the standard curve.

   
   

Pi Concentration = A/V pmol/μl or μM

Where: A is the Pi amount in the reaction from standard curve (in pmol),

V is sample volume added into the reaction well (in μl).