Amplite® Lactose Quantitation Kit
Price | |
Catalog Number | |
Unit Size | |
Quantity |
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Excitation (nm) | 571 |
Emission (nm) | 584 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 12352200 |
Overview | SDSProtocol |
Excitation (nm) 571 | Emission (nm) 584 |
Platform
Absorbance microplate reader
Absorbance | 570 nm |
Recommended plate | Clear bottom |
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Thaw all the kit components to room temperature before starting the experiment.
Prepare and add Lactose Standards and test samples (50 µL)
Prepare and add Enzyme-dye assay reaction mixture (50 µL)
Incubate at 37 °C for 10 - 30 minutes
Monitor Fluorescence intensity at Ex/Em = 540/590 nm or absorbance at OD = 570 nm
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
Prepare stock solution by adding 25 μL of DMSO (Component E) into the vial of Amplite® Red (Component A). The stock solution should be used promptly. Any remaining solution should be aliquoted and refrozen at -20 °C.
Note: Amplite® Red is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM.
Note: Amplite® Red is unstable at high pH (>8.5). The reaction should be performed at pH 7 – 8. The provided assay buffer (pH 7.4) is recommended.
Add 1 mL of assay buffer (Component B) into the vial of horseradish peroxidase (Component C).
Note: The unused HRP solution should be divided into single-use aliquots and stored at -20 °C.
Add 500 µL of assay buffer (Component B) into the vial of Lactose Standard (Component F) to make a 20 mM stock solution. Diluted to 1 mM in assay buffer (10 uL of 20 mM to 190 uL assay buffer).
Note: The unused Lactose Standard stock solution should be stored at -20 °C.
Add 100 µL of assay buffer (Component B) into the vial of enzyme mix (Component D).
Note: The unused enzyme mix stock solution should be divided into single-use aliquots and stored at -20 °C.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/40008
PREPARATION OF WORKING SOLUTION
Prepare the enzyme-dye assay reaction mixture according to the following table, protected from light.
Components | Volume |
Amplite® Red Stock Solution | 25 µL |
HRP Stock Solution | 100 µL |
Enzyme Mix | 100 µL |
Assay Buffer (Component B) | 4.75 µL |
Total Volume | 5 µL |
SAMPLE EXPERIMENTAL PROTOCOL
LS=Sodium Standards (LS1 - LS7, 200 to 3.12 µM for colorimetric analysis and 50 to 0.78 µM for fluorometric analysis); BL=Blank Control; TS=Test Samples
BL | BL | TS | TS |
LS1 | LS1 | ... | ... |
LS2 | LS2 | ... | ... |
LS3 | LS3 | ||
LS4 | LS4 | ||
LS5 | LS5 | ||
LS6 | LS6 | ||
LS7 | LS7 |
Well | Volume | Reagent |
LS1-LS7 | 50 µL | Serial dilutions (according to Table 2) |
BL | 50 µL | Assay Buffer |
TS | 50 µL | Sample |
Prepare Lactose 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 µL of Enzyme-dye reaction mixture to each well of Sodium Standards, blank control, and test samples to make the assay volume 100 µL/well. For a 384-well plate, add 25 µL into each well instead, for a total volume of 50 µL/well.
Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.
Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (cut off at 570 nm) or absorbance with an absorbance microplate reader at OD = 570 nm.
APPENDIX
Add 50 µL of a diluted Lactose Standard stock solution (1 mM) to 200 µL of Assay Buffer (Component B) to make a 200 µM (LS1) dilution. Then perform 1:2 serial dilutions to get serially diluted Standard (LS2 – LS7).
Product Family
Name | Excitation (nm) | Emission (nm) |
Amplite® Ethanol Quantitation Kit | 571 | 584 |
Amplite® Cholesterol Quantitation Kit | 571 | 584 |
Amplite® Choline Quantitation Kit | 571 | 584 |
Images
References
Authors: Ivory, Ruth and Mangan, David and McCleary, Barry V
Journal: Journal of AOAC International (2022): 1617-1624
Authors: Ivory, Ruth and Delaney, Elaine and Mangan, David and McCleary, Barry V
Journal: Journal of AOAC International (2021): 1308-1322
Authors: Wagner, C and Amamcharla, J K and Rao, A and Metzger, L E
Journal: Journal of dairy science (2020): 7585-7597
Authors: Keltanen, Terhi N and Heikman, Pertti K and Muhonen, Leea H and Gunnar, Teemu O and Ojanperä, Ilkka A
Journal: Drug testing and analysis (2019): 1412-1418
Authors: Sun, Jingjing and Yao, Congyu and Wang, Wei and Zhuang, Zhiwei and Liu, Junzhong and Dai, Fangqun and Hao, Jianhua
Journal: Marine drugs (2018)
Authors: Jasti, Lakshmi Swarnalatha and Dola, Sandhya Rani and Fadnavis, Nitin W and Addepally, Uma and Daniels, Siona and Ponrathnam, Surendra
Journal: Enzyme and microbial technology (2014): 67-73
Authors: Condezo-Hoyos, Luis and Mohanty, Indira P and Noratto, Giuliana D
Journal: Journal of dairy science (2014): 2066-70
Authors: Fornera, Sara and Yazawa, Kenjiro and Walde, Peter
Journal: Analytical and bioanalytical chemistry (2011): 2307-10
Authors: Neal, Philip and Cullen, Pauline M
Journal: Paediatric anaesthesia (2004): 279-80
Authors: Jenkins, Daniel M and Delwiche, Michael J
Journal: Biosensors & bioelectronics (2003): 101-7
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