實驗概要
The Aspartate Assay Kit provides a simple, convenient assay to measure aspartate in a variety of samples. In the assay, aspartate is converted to pyruvate which is oxidized with the conversion of a probe into a highly colored (570 nm) and fluorescent (Ex/Em 535/587 nm) species proportional to the amount of aspartate in samples. Aspartate can be quantified in the range between 0.1–10 nmoles/well (2-200 μM).
實驗步驟
1. Reagent Preparation:
1) Aspartate Probe:Ready to use
2)Serum CleanUp Mix, Aspartate Enzyme Mix, Aspartate Substrate Mix, Conversion Mix:Add 220 μl of Aspartate Assay Buffer to each vial respectively and dissolve completely prior to use by pipetting up and down.
2. Assay Protocol:
1) Standard Curve Preparation:
Colorimetric Assay:Dilute the Aspartate Standard to 1.0 mM by adding 10 μl of the 100 mM Aspartate Standard to 990 μl of dH2O, mix well. Add 0, 2, 4, 6, 8, 10 μl into a series of wells. Adjust volume to 50 μl/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of the Aspartate Standard.
2) Fluorometric Assay: Dilute the Aspartate standard to 1 mM as in the colorimetric Assay. Dilute another 10X by taking 100 μl of the standard and adding 900 μl of dH2O, mix well. Add 0, 2, 4, 6, 8, 10 μl into a series of wells. Adjust volume to 50 μl/well with Assay Buffer to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well of the Aspartate Standard.
3) Sample Preparation:
Cell extracts can be used directly in the assay. Serum samples require pretreatment to remove interfering substances: Add 2 μl of the Serum CleanUp Mix to 100 μl serum and incubate 30 minutes at room temperature. Treated serum samples should be deproteinized by centrifuging 10 minutes with a 10kD spin filter. The filtrate (1-30 μl) can be used directly in the assay. Adjust all well volumes to 50 μl with Assay Buffer. Serum aspartate normal range is 0 (undetectable)-25 nmol/ml. Due to the relatively low levels of aspartate in serum, use of the fluorometric assay is recommended.
4) Reaction Mix:
Sample | Sample Control* | |
Aspartate Enzyme Mix | 2 μl | --- |
Conversion Mix | 2 μl | 2 μl |
Aspartate Probe** | 2 μl | 2 μl |
Aspartate Buffer | 34 μl | 36 μl |
Pyruvate in samples can cause background color or fluorescence. This background can be subtracted by performing a sample control in the absence of the Aspartate Enzyme Mix.
In order to reduce background in the fluorometric assay, use 0.5 μl of probe for fluorometric assay per well.
Add 50 μl of the Reaction Mix to each well containing the Standards and Samples.
5) Incubate: For 30 minutes at room temperature protected from light.
6) Read: Measure absorbance (570 nm) or fluorescence (Ex/Em 535/587 nm) in a microplate reader.
7) Calculation: Correct background by subtracting the value of the 0 Aspartate Standard from all readings. Next subtract the value of the Sample Control from the samples.
(Note: The background reading can be significant and must be subtracted from all readings). Plot the Standard curve. Apply the sample readings to the standard curve. Sample aspartate concentrations: C = Sa/Sv = nmol/μl or mM
Where: Sa is the sample amount (in nmol) from standard curve.
Sv is the sample volume (μl) added into the wells.
Aspartate MW: 65.384 g/mol.
