PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit *UV absorption*

Protocol summary

1. Prepare 50 µL of test samples and/or phosphate standards
2. Add 50 µL of working solution
3. Incubate at room temperature for 30 minutes
4. Monitor absorbance at 360 nm

Important notes
Thaw all the four components at room temperature before use.

1. Add 500 µL of ddH₂O to the vial of MESG Substrate (Component B). Mix well by vortexing to get MESG Substrate solution. Note: 250 µl is enough for one plate.
   
   
2. Add 100 µL of ddH₂O to the vial of Purine Nucleoside Phosphorylase (PNP; Component C). Mix well by vortexing to get Purine Nucleoside Phosphorylase solution.
   
   
3. Add the whole volume of MESG Substrate solution and Purine Nucleoside Phosphorylase solution into the bottle of Assay Buffer (Component A) and mix well to get the working solution. Place the working solution on ice. Note: This working solution is stable for at least 4 hours on ice. It is not recommended to freeze the working solution for another assay. To achieve the desirable results, UV-transparent plates or cuvettes are required. Due to the high sensitivity of this assay to Pi, it is extremely important to use Pi-free laboratory ware.

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

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 25 µL of reagent per well instead of 50 µL.
   
   
2. Add 50 µL of working solution to each well of Phosphate standard, blank control, and test samples to make the total assay volume of 100 µL/well. Mix the reagents thoroughly. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate at room temperature for 30 minutes.
   
   
4. Monitor the absorbance with a microplate reader or spectrophotometer at 360 nm. Note: For cuvette assay that requires the total volume larger than 100 µL, multiply the volume of sample and assay reagent proportionally before measuring the absorption.