AspartateAssay

实验概要

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).

实验步骤

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:

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.