Amplite® Colorimetric L-Lactate Assay Kit

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R-phrase	R20, R21, R22
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Overview SDS Protocol

Lactic acid is chiral and has two optical isomers: L-lactic acid and D-lactic acid. Lactate is constantly produced from pyruvate via the enzyme lactate dehydrogenase (LDH) in the process of metabolism and exercise. Monitoring lactate levels is a good way to evaluate the balance between tissue oxygen demand and utilization and is useful when studying cellular and animal physiology. D-lactate is not metabolized by mammals and its elimination from the body depends mainly on renal excretion. D- and L-lactic acid are found in many fermented milk products such as yoghurt and cheese, and also in pickled vegetables, and cured meats and fish. The D- and L-lactic acid (generated by bacteria) is a quality indicator of foods, such as egg, milk, fruit juice and wine. Abnormal high concentration of D-lactate in the blood is usually a reflection of bacterial overgrowth in the gastrointestinal tract. AAT Bioquest's Amplite® Lactate Assay Kits (Cat# 13814 and 13815 for L-lactate assay, and Cat# 13810 and 13811 for D-lactate assay) provide both fluorescence and absorbance-based method for detecting either L-lactate or D-lactate in biological samples such as serum, plasma, urine, as well as in cell culture samples. In the enzyme coupled assay, lactate is proportionally related to NADH, which is specifically monitored by a chromogenic NADH sensor. The signal can easily read by an absorbance microplate reader at the absorbance ratio of ~A575nm/A605nm to increase assay sensitivity. With this Colorimetric Amplite® L-Lactate Assay Kit, we were able to detect as little as 4 µM L-lactate in a 100 µL reaction volume.

Platform

Absorbance microplate reader

Absorbance	575/605 nm
Recommended plate	Clear bottom

Components

Example protocol

AT A GLANCE

Protocol Summary

Prepare L-Lactate standards or test samples (50 µL)
Add L-Lactate working solution (50 µL)
Incubate at room temperature for 30 min - 2 hours
Monitor absorbance ratio increase at A_575nm/A_605nm

Important Thaw one vial of each kit component at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

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.

1. NAD stock solution (100X)

Add 100 µL of H₂O into the vial of NAD (Component C) to make 100X NAD stock solution.

2. L-Lactate standard solution (100 mM)

Add 200 µL of H₂O or 1x PBS buffer into the vial of L-Lactate Standard (Component D) to make 100 mM L-Lactate standard solution.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/13815

L-Lactate standard

Add 10 µL of 100 mM L-Lactate standard solution into 990 µL 1xPBS buffer to generate 1 mM L-Lactate standard solution (SD7). Take 1 mM L-Lactate standard solution (SD7) and perform 1:3 serial dilutions in 1xPBS buffer to get serially diluted L-Lactate standards (SD6 - SD1). Note: Diluted L-Lactate standard solution is unstable, and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Probe (Component A), and mix well.
Add 50 µL of 100X NAD stock solution into the bottle of Component A+B and mix well to make L-Lactate working solution.
Note This L-Lactate working solution is enough for one 96-well plate. It is unstable and should be used promptly within 2 hours. Avoid exposure to light.
Note Alternatively, one can make a 50X of L-Lactate Enzyme stock solution by adding 100 μL of H2O into the bottle of Enzyme Mix (Component A), and then prepare the L-Lactate working solution by mix the stock solution with Assay Buffer (Component B) and 100X NAD stock solution proportionally.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of L-Lactate standards and test samples in a white clear bottom 96-well microplate. SD= L-Lactate Standards (SD1 - SD7, 1 to 1000 µM), BL=Blank Control, TS=Test Samples.

BL	BL	TS	TS
SD1	SD1	...	...
SD2	SD2	...	...
SD3	SD3
SD4	SD4
SD5	SD5
SD6	SD6
SD7	SD7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
SD1 - SD7	50 µL	Serial Dilutions (1 to 1000 µM)
BL	50 µL	Dilution Buffer
TS	50 µL	test sample

Prepare L-Lactate standards (SD), 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 µL of L-Lactate working solution to each well of L-Lactate standard, blank control, and test samples to make the total L-Lactate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of L-Lactate working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.
Monitor the absorbance ratio increase with an absorbance plate reader at A_575nm/A_605nm.

Images

Figure 1. L-Lactate dose response was measured with Amplite® Colorimetric L-Lactate Assay Kit in a 96-well white clear bottom plate using a SpectraMax Plus (Molecular Devices) microplate reader.

Citations

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