Amplite® Fluorimetric Lead (II) Ion Quantitation Kit

Lead is a highly poisonous metal to human beings, especially young children. Lead interferes with cell signaling and gene expression, causing severe damages to the brain, liver, kidney, and even death. Because of its long history in mining and smelting, as well as widespread use in battery, paint and gasoline, lead pollution in soil and groundwater has been one of the most serious environmental problems. Amplite® Fluorimetric Lead (II) Ion Quantitation Kit provides a robust method for detecting lead (II) ion in solution. It uses Lead Green™, a selective and sensitive green fluorescence probe that can be easily monitored with a fluorescence microplate reader (Ex/Em = 490/530 nm). The Amplite® Fluorimetric Lead (II) Ion Quantitation Kit can be performed in a convenient 96-well or 384-well microplate format and easily adapted to automation with no separation steps required. The assay can be completed within 30 minutes. With the Amplite® Fluorimetric Lead (II) Ion Quantitation Kit, as little as 4µM lead (II) ion was detected.

Example protocol

AT A GLANCE

Protocol summary

Prepare and add lead standards or test samples (50 uL)
Prepare and add Lead Green working solution (50 uL)
Incubate at room temperature for 10-30 minutes
Monitor fluorescence intensity at Ex/Em = 490/530 nm

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

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Lead Green stock solution (200X):
Add 50 uL of DMSO (Component D) into the vial of Lead Green (Component A) to make Lead Green stock solution (200X). Note: Store the unused Lead Green stock solution at -20 °C in single use aliquots.

PREPARATION OF STANDARD SOLUTION

Lead standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/19007

Add 10 uL of 100 mM Lead (II) Standard solution (Component C) to 990 uL H₂O to generate 1 mM Lead standard solution. Take 1 mM Lead (II) standard solution to perform 1:3 serial dilutions by H₂O to get serially diluted Lead (II) standards ranging from 0 to 1 mM.

PREPARATION OF WORKING SOLUTION

Lead Green working solution:
Add 25 uL Lead Green stock solution (200X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.025 mL. Note: Keep away from Light. Note: Lead Green working solution should be used promptly.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of lead standards and test samples in a solid black 96-well microplate. LS = Lead standard (LS1-LS7, 1000 to 1.4 uM); BL = blank control; TS = test sample.

BL	BL	TS	TS
LS1	LS1	...	...
LS2	LS2	...	...
LS3	LS3
LS4	LS4
LS5	LS5
LS6	LS6
LS7	LS7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
LS1-LS7	50 uL	serial dilution (1000 to 1.34 uM)
BL	50 uL	H₂O
TS	50 uL	test sample

Prepare Lead Standards (LS), 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.
Add 50 uL of Lead Green working solution to each well of Lead Standard, blank control, and test samples to make the total Lead assay volume of 100 uL/well. For a 384-well plate, add 25 uL of working solution into each well instead for a total volume of 50 uL/well.
Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.
Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 490/530 nm.

Citations

View all 20 citations: Citation Explorer

Lead toxicity from retained bullet fragments: A systematic review and meta-analysis
Authors: Apte, A., Bradford, K., Dente, C., Smith, R. N.
Journal: J Trauma Acute Care Surg (2019): 707-716

Toxicodynamics of Lead, Cadmium, Mercury and Arsenic- induced kidney toxicity and treatment strategy: A mini review
Authors: Rana, M. N., Tangpong, J., Rahman, M. M.
Journal: Toxicol Rep (2018): 704-713

A critical review on speciation, mobilization and toxicity of lead in soil-microbe-plant system and bioremediation strategies
Authors: Kushwaha, A., Hans, N., Kumar, S., Rani, R.
Journal: Ecotoxicol Environ Saf (2018): 1035-1045

Severe Systemic Lead Toxicity Resulting From Extra-Articular Retained Shrapnel Presenting as Jaundice and Hepatitis: A Case Report and Review of the Literature
Authors: Grasso, I. A., Blattner, M. R., Short, T., Downs, J. W.
Journal: Mil Med (2017): e1843-e1848

Retained Lumbar Bullet: A Case Report of Chronic Lead Toxicity and Review of the Literature
Authors: Bustamante, N. D., Macias-Konstantopoulos, W. L.
Journal: J Emerg Med (2016): 45-9

Page updated on December 3, 2024

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