Oxaloacetate Colorimetric/Fluorometric Assay Kit

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Reaktivität
Chemical
1
Detektionsbereich
0.1-10 nM
Untere Nachweisgrenze
0.1 nM
Applikation
Biochemical Assay (BCA)
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Proben Cell Culture Supernatant, Plasma, Serum, Tissue Samples
Nachweismethode Fluorometric, Colorimetric
Spezifität Oxaloacetate Assay Kit provides a simple, sensitive and rapid means of quantifying OAA in a variety of samples. In the assay, OAA is converted to pyruvate which is utilized to convert a nearly colorless probe to an intensely colored (lambda max = 570nm) and fluorescent (E x /E m = 535/587nm) product. The Oxaloacetate Assay Kit can detect 0.1-10nmol (2-200 ?M) of OAA.
Produktmerkmale Oxaloacetate Assay Kit: Colorimetric & Fluorometric Assay for Quantifying Oxaloacetate (OAA) in a variety of Samples such as Tissues, Cells etc. within 40 min. Rapid, Convenient & Reliable.
Bestandteile OAA Assay Buffer
OAA Probe ( in DMSO)
OAA Enzyme Mix
Developer
OAA Standard (10 μmol)
Substanzklasse Chemical
Hintergrund Oxaloacetate (OAA, HOOC-CO-CH₂-COOH) is a TCA cycle intermediate. It precedes citrate which is formed by the transfer of an acetyl group to OAA. OAA is formed by the deamidation of aspartate or condensation of CO2 with pyruvate or PEP. Since mammals do not possess the enzymatic machinery to form TCA cycle intermediates from acetyl CoA, OAA is one of the anaplerotic entry points via pyruvate and pyruvate carboxykinase.
Applikationshinweise The Oxaloacetate Assay Kit can detect 0.1-10nmol (2-200 μM) of OAA.
Kommentare

Further details regarding sample type: Cell and tissue culture supernatants, urine, plasma and serum, as well as many other biological fluids

Testdauer < 1 h
Protokoll 1. Standard Curve Preparations: Colorimetric Assay: Dilute OAA Standard to1 nM/µL by adding 10 µL of the standard to 990 µL of dH2O, mix well. Add 0, 2, 4, 6, 8, 10 µL into a series of wells on a 96 well plate. Adjust volume to 50 µL/well with OAA Assay Buffer to generate 0, 2, 4, 6, 8, 10 nM/well of the standard. Fluorometric Assay: Dilute OAA standard to 0.1 nM/µL by adding 10 ul of the standard to 990 µL of dH2O, mix well, then further dilute by adding 10 µL to 90 µL of dH2O. Add 0, 2, 4, 6, 8, 10 µL into a series of standards well on a 96-well plate. Adjust volume to 50 µL/well to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nM/well OAA Standard.
2. Sample Preparation: Tissue (20 mg) or cells (2 x 10^6 ) should be rapidly homogenized with 100 µL OAA Assay Buffer. Centrifuge 15000g for 10 min to remove insoluble materials. Enzymes in samples may interfere with the assay. We suggest deproteinizing samples using a perchloric acid/KOH protocol or 10 kd molecular weight cut off spin columns. Add 1-50 µL samples into duplicate wells of a 96-well plate and bring volume to 50 µL with Assay Buffer. We suggest testing several doses of your samples to ensure readings are within the standard curve range.
3. Develop: Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 µL Reaction Mix containing: Colorimetric Assay Fluorometric Assay Sample Sample Control Sample Sample Control OAA Assay Buffer 44 µL 46 µL 44 µL 46 µL OAA Enzyme Mix 2 µL ---- 2 µL ---- OAA Developer 2 µL 2 µL 2 µL 2 µL OAA Probe ** 2 µL 2 µL 2 µL 2 µL Notes: *Pyruvate in samples can cause background color or fluorescence. This background can be subtracted by performing a sample control in the absence of the OAA Enzyme Mix. **In the fluorometric assay, dilute an aliquot of probe 10x with DMSO to reduce fluorescent background. Add 50 µL of the Reaction Mix to each well containing the OAA Standard and test samples.
4. Incubate for 30 minutes at room temperature, protect from light.
5. Measure OD at 570 nm or fluorescence E x /E m at 535/587nm with a 96 well plate reader.
Ergebnisberechnung

Calculation: Correct background by subtracting the value of the 0 OAA 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 sample readings). Plot the standard curve. Apply corrected sample readings to the standard curve to get OAA amount in the sample wells. The OAA concentrations in the test samples: C = Ay/Sv (nM/µL, or μM/mL, or mM) Where: Ay is the amount of OAA(nmol) in your sample from the standard curve. Sv is the sample volume (µL) added to the sample well. Oxaloacetic Acid molecular weight: 132.07 g/M Oxaloacetate standard curve generated following the kit protocol.

Beschränkungen Nur für Forschungszwecke einsetzbar
Lagerung -20 °C
Haltbarkeit 12 months
Produkt verwendet in: Chang, Jeon, Gu, Pack, Jin: "Conversion of carbon dioxide to oxaloacetate using integrated carbonic anhydrase and phosphoenolpyruvate carboxylase." in: Bioprocess and biosystems engineering, 2013 (PubMed).

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