PhosphoWorks™ Colorimetric Phosphate Assay Kit *Blue Color*

Protocol summary

1. Prepare test samples or Phosphate standards (80 µL)
2. Add MG Plus™ Reagent (Component B) (20 µL)
3. Incubate at room temperature for 10 - 40 minutes
4. Monitor absorbance at 600 - 660 nm or spectrophotometer

Important notes
Phosphate-containing buffers should be avoided when preparing the samples. To achieve the best results, it is strongly recommend to use clear microplates or cuvettes. Thaw all the kit components at room temperature before starting the experiment.

Table 1. Layout of Phosphate standards and test samples in a clear 96-well microplate. PS=Phosphate Standards (PS1 - PS7, 0.78 to 50 µM), BL=Blank Control, TS=Test Samples. 

Table 2. Reagent composition for each well.

1. Prepare Phosphate standards (PS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 40 µL of reagent per well instead of 80 µL.
   
   
2. Shake MG Plus™ Reagent (Component B) well before use.
   
   
3. Add 20 µL of MG Plus™ Reagent (Component B) to each well of Phosphate standard, blank control, and test samples to make the total Phosphate assay volume of 100 µL/well. Mix the reagents thoroughly. For a 384-well plate, add 10 µL of MG Plus™ Reagent (Component B) into each well instead, for a total volume of 50 µL/well.
   
   
4. A blue-green color will develop in the phosphate-containing wells in 10 to 40 minutes. Monitor absorbance with an absorbance microplate reader at 600 - 660 nm or a spectrophotometer. Note: At high phosphate concentration (>100 µM), precipitates may form. Dilute your samples and redo the assays. Note: For cuvette assay that requires the total volume larger than 100 µL, either multiple the volume of sample and MG Plus™ Reagent (Component B) proportionally or dilute the final reaction mixture with 1 M H₂SO₄ or 1 M HCl before measuring the absorption.