Phosphate Colorimetric Assay Kit

(Catalog #K410-500; 500 assays; Store Kit at Room Temp.)

I. Introduction:

Phosphate is one of the most important of the inorganic 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. Phosphate is also of great importance in mineralization processes and is a primary stimulus of algal blooms frequently found in bodies of fresh water, due to run-off from areas of high fertilizer use. The newly designed Phosphate Colorimetric Assay Kit provides an easy, quick and sensitive means of assessing phosphate over a wide range of concentrations. The assay utilizes a proprietary formulation of malachite green and ammonium molybdate which forms a chromogenic complex with phosphate ion giving an intense absorption band around 650 nm. Phosphate concentrations between 1 µM and 1 mM, with a lower limit of detection of approximately 0.1 nmol, can be directly determined. The Phosphate Colorimetric Assay Kit provides 500 assays using microtiter plates or 100 assays using 1 ml cuvettes. 

II. Kit Contents:

 

 

 

III. Reconstitution of Reagents:

Phosphate Reagent: Ready to use as supplied and may be kept at room temperature. There may be a small amount of precipitate visible which doesn’t affect the assay.

IV. Assay Protocol:

1. 
   

   Phosphate Standard Curve: Dilute 10 µl of the 10 mM Phosphate Standard to 990 µl dH₂O, mix well to generate 100 µM working Phosphate Standard. Add 0, 10, 20, 30, 40, 50 µl of the 100 M working Phosphate Standard to individual wells. Adjust the volume to 200 µl with dH₂O to generate 0, 1, 2, 3, 4, 5 nmol of Phosphate standard.

   
   

2. Preparation of sample: No sample pretreatment is necessary. Add between 0-200 µl of sample for the assays and bring the well volume to 200 µl with distilled water. If the approximate phosphate concentration is not known, we recommend widely different sample volumes (1 – 10 – 100 µl) be tested.

The absorbance of samples should be in the linear range of the standard curve (0-5 nmol/well). If they fall outside of this range, samples should be diluted and rerun or smaller sample volumes be used. The detection limit of the assay is approximately 0.1 nmol per well (1 µM) of Phosphate.

3. Reaction:

1. 
   

   Add 30 µl Phosphate Reagent to all standard and sample wells, mix well.

   
   


2. 
   

   Cover the plate and incubate at room temperature for 30 minutes.

   
   


3. 
   

   Read the absorbance at 650 nm using a plate reader. The color is stable for several hours.

   
   

 

 

 

 

4. Protocol for using 1.0 ml cuvettes: Increase all reaction components 5x when using 1 ml cuvettes. The 1 ml total reaction mixture will contain 0-25 nmol phosphate (0-500 µl), 150 µl of Phosphate Reagent and made up to 1.0 ml with distilled water. Incubate at room temperature for 30 min then read at 650 nm.

5. Calculations:

1. 
   

   Plot standard curve: Plot absorbance at 650 nm as a function of Phosphate concentration.

   
   


2. 
   

   Determine sample Phosphate concentration:

   
   

 

Phosphate concentration =

OR

= nmol/µl or mM Phosphate

Where Sa is the sample amount (in nmoles) read from the standard curve.

Sv is the sample volume (undiluted) added to the wells.

Caution: Many laboratory detergents contain high amounts of phosphates which can adhere to cleaned glassware. It is highly recommended to use disposable plastic labware for all samples, standards and reagents to avoid contamination.