Phosphatidylcholine 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 Phosphatidylcholine Assay Kit is a simple convenient means of measuring Phosphatidylcholine in a variety of biological samples. The kit utilizes an enzyme-coupled assay in which PC is hydrolyzed, releasing choline which is subsequently oxidized resulting in development of the OxiRed probe to generate fluorescence (Ex/Em = 535/587 nm) and absorbance (O.D. = 570 nm). The kit measures PC in the range of 0.1 to 10 nmol per sample. PC is present in serum at approx. 0.2-2.5 mM (approx. 50-200?mg/dL).
Produktmerkmale Phosphatidylcholine Assay Kit: Colorimetric & Fluorometric Assay to Measure Phosphatidylcholine (PC) in a variety of Biological Samples within 40 min. Simple & Convenient.
Bestandteile PC Assay Buffer
OxiRed Probe
PC Hydrolysis Enzyme
PC Development Mix
PC Standard (10 μmol)
Substanzklasse Chemical
Hintergrund Phosphatidylcholine (PC) is a phospholipid which incorporates choline as the headgroup of the lipid. PC is a major constituent of biological membranes and is involved in cell signaling through release of choline by phospholipase D leaving the second messenger phosphatidic acid. Phosphatidylcholine Assay Kit is a simple convenient means of measuring Phosphatidylcholine in a variety of biological samples. The kit utilizes an enzyme-coupled assay in which PC is hydrolyzed, releasing choline which is subsequently oxidized resulting in development of the OxiRed probe to generate fluorescence (Ex/Em 535 nm 587 nm) and absorbance (570 nm).
Applikationshinweise BioVision's Phosphatidylcholine kit measures PC in the range of 0.1 to 10 nmol per sample.
Kommentare

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

Testdauer < 1 h
Protokoll 1. Standard Curve: For the Colorimetric Assay: Dilute 10 µL of the 100 mM PC Standard with 990 µL dH2O to generate 1 mM Standard Phosphatidylcholine. Add 0, 2, 4, 6, 8, 10 µL of the diluted PC Standard into a 96-well plate to generate 0, 2, 4, 6, 8, 10 nM/well Standard. Bring the volume to 50 µL with Assay Buffer. For the Fluorometric Assay: Dilute the standard to 0.1 mM (0.1 nM/µL), then follow the same protocol as colorimetric assay, to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nM/well of the Standard.
2. Sample Preparation: Liquid sample can be tested directly. Tissue or cells can be extracted using 4-10 volume of Assay Buffer. Centrifuge at 15000 rpm for 10 min. Transfer the extract to a fresh tube. Add samples to sample wells in a 96-well plate and bring the volume to 50 µL/well with Assay Buffer. We suggest testing several doses of your sample to make sure the readings are within the standard curve range.
3. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 50 µL Reaction Mix containing: For Phosphatidylcholine Measurement For Background Control * 44 µL Assay Buffer 46 µL Assay Buffer 2 µL PC Converting Enzyme ---------------------- 2 µL Development Mix 2 µL Development Mix 2 µL PC Probe** 2 µL PC Probe *Choline can generate background in samples. If Choline is suspected to be in your sample, perform a background control without the PC Converting Enzyme. Subtract the background readings from sample readings. ** For the fluorescent assay, dilute the probe 10X to reduce background reading. Add 50 µL of the Reaction Mix to each well containing the PC Standard and test samples. Mix well. Incubate the reaction for 30 min at room temperature, protect from light.
4. Measure O.D. at 570 nm or fluorescence at Ex/Em 535/587 nm in a microplate reader.
Ergebnisberechnung

Calculation: Correct background by subtracting the value derived from the 0 PC control from all sample and standard readings (The background reading can be significant and must be subtracted from sample readings). Plot PC standard curve. Apply sample readings to the standard curve. PC concentrations of the test samples can then be calculated: C = S a /S v (nM/µL, μM/mL or mM) Where S a is the PC content of unknown samples (in nmol) from standard curve, S v is sample volume (µL) added into the assay wells. Phosphatidylcholine avg molecular weight is 768 g/mol.

Beschränkungen Nur für Forschungszwecke einsetzbar
Lagerung -20 °C
Haltbarkeit 12 months
Produkt verwendet in: Yuyama, Sun, Sakai, Mitsutake, Okada, Tahara, Furukawa, Fujitani, Shinohara, Igarashi: "Decreased amyloid-β pathologies by intracerebral loading of glycosphingolipid-enriched exosomes in Alzheimer model mice." in: The Journal of biological chemistry, Vol. 289, Issue 35, pp. 24488-98, 2014 (PubMed).

Mitsutake, Zama, Yokota, Yoshida, Tanaka, Mitsui, Ikawa, Okabe, Tanaka, Yamashita, Takemoto, Okazaki, Watanabe, Igarashi: "Dynamic modification of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and type 2 diabetes." in: The Journal of biological chemistry, Vol. 286, Issue 32, pp. 28544-55, 2011 (PubMed).

Li, Na, Lee, Lee, Paik: "Contribution of sams-1 and pmt-1 to lipid homoeostasis in adult Caenorhabditis elegans." in: Journal of biochemistry, Vol. 149, Issue 5, pp. 529-38, 2011 (PubMed).

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