AAT Bioquest
Product Technical Information Sheet
Last Updated September 2014
Amplite™ Fluorimetric Aspartate (Aspartic Acid) Assay Kit
*Red Fluorescence*
Ordering Information
Cat#: 13827 (200 Assays)
Storage Conditions
Instrument Platform
Keep in freezer and protect from light
Fluorescence microplate readers
Introduction
Aspartate (or Aspartic acid) is a negatively charged, polar amino acid. Aspartate is involved in the control point of
pyrimidine biosynthesis, in transamination reactions, interconversions with asparagine, in the metabolic pathway leading to
AMP, in the urea cycle, and is a precursor to homoserine, threonine, isoleucine, and methionine. It is also involved in the
malate aspartate shuttle. Amplite™ Fluorimetric Aspartate Assay Kit offers a sensitive fluorescent assay for quantifying
aspartate in biological samples. Aspartate is converted to pyruvate that generates hydrogen peroxide through an enzyme
coupled reaction. The amount of hydrogen peroxide generated by aspartate is monitored with Amplite™ Red substrate for
quantifying aspartate by a fluorescence microplate reader at Ex/Em = 540/590 nm.
Kit Components
Components
Component A: Amplite™ Red Substrate (light sensitive)
Component B: Enzyme Mix
Component C: Assay Buffer
Component D: Conversion Mix
Component E: Aspartate Standard
Component F: DMSO
Amount
1 vial
2 bottles (lyophilized powder)
1 bottle (10 mL)
2 vials (lyophilized powder)
1 vial (lyophilized powder)
1 vial (100 µL)
Assay Protocol for One 96-Well Plate
Brief Summary
Prepare test samples (50 µL) along with serially diluted aspartate standards (50 µL)  Add equal
volume of Assay Mixture (50 µL)  Incubate at 37ºC for 20-30 minutes
 Monitor fluorescence intensity at Ex/Em = 540/590 nm
Note: Thaw kit components at room temperature before use. To achieve the best results, it’s recommended to use the black
plates.
1. Prepare serially diluted aspartate standards and test samples:
1.1 Add 100uL of dd H2O into Aspartate Standard vial (Component E) to make 10 mM aspartate stock solution.
1.2 Prepare aspartate standard dilutions: Add 10 μL of 10 mM aspartate standard (from step 1.1) into 990 μL of 1×PBS
buffer to get 100 µM aspartate solution. Then perform 1:3 serial dilutions in 1×PBS buffer to get approximately 30, 10,
3, 1, 0.3, and 0.1 μM serially diluted aspartate standards.
1.3 Add aspartate containing samples and serially diluted aspartate standards into a solid black 96-well microplate according
to Tables 1.
Table 1 Layout of aspartate standards and test samples in a solid black 96-well microplate
BL
BL
TS
TS
….
….
ASP 1
ASP 1
….
….
….
….
ASP 2
ASP 2
ASP 3
ASP 3
ASP 4
ASP 4
ASP 5
ASP 5
©2014 by AAT Bioquest, Inc., 520 Mercury Drive, Sunnyvale, CA 94085. Tel: 408-733-1055
Ordering: sales@aatbio.com; 800-990-8053 or 408-733-1055; Fax: 408-733-1304
Technical Support: support@aatbio.com; 408-733-1055
AAT Bioquest
Product Technical Information Sheet
Last Updated September 2014
ASP 6
ASP 6
ASP 7
ASP 7
Note 1: ASP= Aspartate Standard, BL=Blank Control (1×PBS buffer), TS=Test Sample.
Note 2: Add the serial dilutions of aspartate standards from 0.1 μM to 100 μM into wells from ASP 1 to ASP 7.
2. Prepare Aspartate Assay Mixture:
2.1 Make Amplite™ Red substrate stock solution: Add 50 μL of DMSO (Component F) into Amplite™ Red substrate
(Component A) to make 200X Amplite™ Red substrate stock solution.
2.2 Make Conversion Mix stock solution: Add 50 μL of dd H2O into Conversion Mix (Component D) to make 100X
Conversion Mix stock solution.
2.3 Make Assay Mixture: Add 5mL Assay Buffer (Component C) into one bottle of Enzyme Mix (Component B) mix well,
and then transfer 25μL of 200X Amplite™ Red substrate stock solution (from Step 2.1), 50 μL of 100X Conversion Mix
stock solution (from Step 2.2 into the bottle, and mix well.
Note 1: The assay mixture is not stable, use it promptly, and avoid direct exposure to light.
Note 2: Store unused 200X Amplite™ Red stock solution at -20 oC, avoid light and repeated freeze-thaw cycles.
3. Run aspartate assay:
3.1 Add 50 μL of Assay Mixture (from Step 2.3) into each well of aspartate standard, blank control, and test samples (see
Step 1.3) to make the total aspartate assay volume of 100 µL/well.
Note 1: For a 384-well plate, add 25 μL of sample, 25 μL of Assay mixture into each well.
Note 2: Run the aspartate assay at pH 6.5 to 7.0.
3.2 Incubate the reaction mixture at 37 ºC for 20-30 minutes.
3.3 Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em=540/590 nm (cut off: 570 nm).
Data Analysis
The fluorescence reading in blank wells (with 1X PBS buffer only) is used as a control, and is subtracted from the values
of those wells with the aspartate standards and test samples. The standard curve of aspartate is shown in Figure 1. Calculate the
aspartate concentrations of the samples according to the standard curve.
Figure1. Aspartate dose response was measured with Amplite™ Fluorimetric Aspartate Assay Kit (Cat#13827) on a solid black
96-well plate using a Gemini microplate reader (Molecular Devices). As low as 0.4 µM aspartate can be detected with 20-30 min
incubation. (Note: The fluorescence background increases with time, thus it is important to subtract the fluorescence intensity
value of the blank wells for each data point).
References
1.
2.
Azevedo, RA (2002) Analysis of the aspartic acid metabolic pathway using mutant genes. Amino Acids 22: pp. 217-230.
Muhitch, MJ (1997) Effects of overexpressing E. coli threonine synthase in tobacco (Nicotiana tabacum) suspension
culture cells on free amino acid levels, aspartate pathway enzyme activities and uptake of aspartate into cells. J Plant
Physiol 150: pp. 16-22
©2014 by AAT Bioquest, Inc., 520 Mercury Drive, Sunnyvale, CA 94085. Tel: 408-733-1055
Ordering: sales@aatbio.com; 800-990-8053 or 408-733-1055; Fax: 408-733-1304
Technical Support: support@aatbio.com; 408-733-1055