Amplite® Colorimetric L-Alanine Assay Kit
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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 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
Platform
Absorbance microplate reader
Absorbance | 575 nm |
Recommended plate | Clear bottom |
Components
Example protocol
AT A GLANCE
- Prepare L-Alanine standards or test samples (50 µL)
- Add L-Alanine working solution (50 µL)
Incubate at 37°C for 1 hour to 2 hours
- Monitor absorbance intensity at 575 nm
To achieve the best result, it is strongly recommended to use the white clear plates. Thaw kit components at room temperature before use.
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
Add 50 µL of DMSO (Component E) into Quest Fluor™ L-Alanine Sensor (Component A) to make 200X Quest Fluor™ L-Alanine Sensor stock solution.
Add 10 µL of 100 mM L-Alanine Standard (Component D) into 490 µL of PBS (pH 7.0) to get 2 mM L-Alanine standard solution. Add 7.5 µL of 100 mM L-Alanine Standard (Component D) into 492.5 µL of PBS (pH 7.0) to get 1.5 mM L-Alanine standard solution.
PREPARATION OF WORKING SOLUTION
Use 2 mM L-Alanine standard stock solution and perform 1:2 serial dilutions in PBS to get serially diluted L-Alanine standards (2000, 1000, 500, 250, 125 uM). Use 1.5 mM L-Alanine standard stock solution and perform 1:2 serial dilutions in PBS to get serially diluted L-Alanine standards (1500, 750 uM).
Add 5 mL Assay Buffer (Component C) into one Enzyme Mix1 bottle (Component B1) and mix well.
Add 100 μL of ddH2O into one Enzyme Mix2 vial (Component B2) and mix well.
Transfer entire vial (100 μL) of Enzyme Mix2 and 25 uL of 200X L-Alanine Sensor stock solution into the Enzyme Mix1 bottle and mix well to make L-Alanine working solution. Note: L-Alanine working solution is not stable - use it promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of L-Alanine standards and test samples in a white clear 96-well microplate. AS= L-Alanine Standard (AS1 - AS7, 125 to 2000 µM), BL=Blank Control, TS=Test Sample.
BL | BL | TS | TS |
AS1 | AS1 | ... | ... |
AS2 | AS2 | ... | ... |
AS3 | AS3 | ||
AS4 | AS4 | ||
AS5 | AS5 | ||
AS6 | AS6 | ||
AS7 | AS7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
AS1 - AS7 | 50 µL | Serial Dilutions (125 to 2000 µM) |
BL | 50 µL | PBS |
TS | 50 µL | test sample |
- Prepare L-Alanine standards (AS), 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-Alanine working solution to each well of L-Alanine standard, blank control, and test samples to make the total L-Alanine assay volume of 100 µL/well. For a 384-well plate, add 25 µL of L-Alanine working solution into each well instead, for a total volume of 50 µL/well. Note: Run the L-Alanine assay at pH 6.5 to 7.0.
Incubate the reaction at 37°C for 1 hour to 2 hours.
- Monitor the absorbance increase with an absorbance plate reader at 575 nm.
Images
Citations
Authors: Skibbe, K and Brethack, AK and S{\"u}nderhauf, A and Ragab, M and Raschdorf, A and Hicken, M and Schlichting, H and Preira, J and Brandt, J and Castven, D and others,
Journal: (2021)
Authors: Kappler, Matthias and Pabst, Ulrike and Weinholdt, Claus and Taubert, Helge and Rot, Swetlana and Kaune, Tom and Kotrba, Johanna and Porsch, Martin and G{\"u}ttler, Antje and Bache, Matthias and others,
Journal: International journal of molecular sciences (2019): 4742
References
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Journal: J Phys Chem B (2011): 15339
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Journal: J Agric Food Chem (2010): 120
Authors: Wysocka M, Lesner A, Guzow K, Kulczycka J, Legowska A, Wiczk W, Rolka K.
Journal: Anal Chem (2010): 3883
Authors: Schwimmer JB, Dunn W, Norman GJ, Pardee PE, Middleton MS, Kerkar N, Sirlin CB.
Journal: Gastroenterology (2010): 1357
Authors: Huang FR, Zhang J, Luo YH, Li SP, Zheng SF, Chen XD.
Journal: Guang Pu Xue Yu Guang Pu Fen Xi (2010): 2620
Authors: Song MJ, Yun DH, Hong SI.
Journal: Biosci Biotechnol Biochem (2009): 474
Authors: Glinghammar B, Rafter I, Lindstrom AK, Hedberg JJ, Andersson HB, Lindblom P, Berg AL, Cotgreave I.
Journal: Int J Mol Med (2009): 621
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