Amplite® Fluorimetric Zinc Ion Quantitation Kit

Protocol summary

1. Test samples (50 µL) or Zn²⁺ Standard
2. Add Zinc Probe Reagent (50 µL)
3. Incubate at room temperature for 5 - 10 minutes
4. Read fluorescence at Ex/Em= 485/525 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

1. Zinc standard solution (1 mM):
Add 10 µL of 100 mM ZnCl₂ Standard (Component C) into 990 µL Assay Buffer (Component B) to get 1 mM ZnCl₂ standard solution.

Add 25 µL of Metal Fluor™ Zn 520 (Component A) into 5 mL Assay Buffer (Component B) to make Zn working solution.

Table 1. Layout of ZnCl₂ standards and test samples in a solid black 96-well microplate. Zn = Zinc standard (Zn1 - Zn7, 0.1 to 100 µM); BL = blank control; TS = test sample.

Table 2. Reagent compotition for each well.

1. Dilute the test sample to a 5 - 100 µM range with Assay Buffer (Component B).
   
   
2. Prepare ZnCl₂ standards (Zn), blank controls (BL), and test samples (TS) according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   
3. Add 50 µL of Zinc working solution into each well of ZnCl₂ standard, blank control, and test samples to make the total ZnCl₂ assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Zinc assay buffer into each well instead, for a total volume of 50 µL/well.
   
   
4. Incubate the reaction for 5 - 10 minutes at room temperature, protected from light.
   
   
5. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 485/525 nm.