Serum Triglyceride Quantification Kit (Colorimetric)

Produktnummer ABIN2345058

Kurzübersicht für Serum Triglyceride Quantification Kit (Colorimetric) (ABIN2345058)

Nachweismethode: Colorimetric

Applikation: Quantification (Q)

Proben: Serum, Plasma

Produktdetails

Produktmerkmale: Serum Triglyceride Quantification Kit measures triglyceride concentration in serum, plasma, and lysates by a coupled enzymatic reaction system. First, lipase hydrolyzes the triglyceride ester bond, yielding glycerol. The glycerol is then phosphorylated and oxidized, producing hydrogen peroxide which reacts with the kit's Colorimetric Probe (absorbance maxima of 570 nm). The Serum Triglyceride Quantification Kit is a simple, colorimetric assay that quantitatively measures the amount of triglyceride in plasma, serum, and lysates in a 96-well microtiter plate format. Each kit provides sufficient reagents to perform up to 100 assays, including blanks, triglyceride standards, free glycerol controls and unknown samples. The kit contains a triglyceride standard and has a detection sensitivity limit of ~10 μM (1 mg/dL).

Bestandteile:

1. Triglyceride Standard : One 200 μL vial (equivalent to 20,000 mg/dL triglyceride mixture with average MW of 873).
2. 10X Assay Buffer : One 1.5 mL vial. 2
3. 10X Lipase Solution : One 1 mL vial.
4. 5X Enzyme Mixture : Four 525 μL vials.
5. 200X Colorimetric Probe : One 55 μL amber vial.

Benötigtes Material:

1. 96-well microtiter plate
2. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
3. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
4. Multichannel micropipette reservoir
5. Microplate reader capable of reading at 570 nm

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Kommentare:

• Measure triglyceride concentration in serum, plasma, and lysates by a coupled enzymatic reaction system
• Simple assay that quantitatively measures the amount of triglyceride in a 96-well microtiter plate format
• Kits include triglyceride standards and free glycerol controls

Aufbereitung der Reagenzien:

• Triglyceride Standard, 10X Assay Buffer, 10X Lipase Solution, and 5X Enzyme Mixture should be thawed/maintained at 4 °C during assay preparation. All are stable for 1 week at 4 °C. For longer term storage, each should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws.
• 200X Colorimetric Probe should be thawed/maintained at room temperature during assay preparation. Any unused material should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws.

Aufbereitung der Proben:

• Plasma: Collect blood with an anticoagulant such as heparin, citrate or EDTA and mix by inversion. Centrifuge the blood at 1000 x g at 4 °C for 10 minutes. Collect plasma supernatant without disturbing the white buffy layer. Sample should be tested immediately or frozen at -80 °C for storage. Plasma must be diluted before assaying (1:10 to 1:40 in PBS). Normal triglyceride levels in human plasma are considered less than 150 mg/dL, however, very high levels can exceed 500 mg/dL.
• Serum: Collect blood in a tube with no anticoagulant. Allow the blood to clot at room temperature for 30 minutes. Centrifuge at 2500 x g for 20 minutes. Remove the yellow serum supernatant without disturbing the white buffy layer. Samples should be tested immediately or frozen at -80 °C for storage. Serum must be diluted before assaying (1:10 to 1:40 in PBS). Normal triglyceride levels in human serum are considered less than 150 mg/dL, however, very high levels can exceed 500 mg/dL.
• Cell Lysates: Collect 10 x 106 cells by centrifugation at 1000 x g for 10 minutes. Discard the supernatant and resuspend in 1 mL of cold PBS containing 1 % Triton X-100. Homogenize or sonicate the cell suspension. Centrifuge at 10000 x g for 10 minutes at 4 °C. Carefully collect the supernatant and store on ice for immediate use. For longer term storage, freeze the lysate at -80 °C for up to 1 month. Cell lysates must be further diluted before assaying (1:5 or greater).
• Tissue Samples: Weigh out 200 mg of tissue and mince into small pieces. Homogenize the minced tissue in 1 mL of cold PBS containing 1 % Triton X-100. Centrifuge at 1000 x g for 10 minutes at 4 °C. Carefully collect the supernatant and store on ice for immediate use. For longer term storage, freeze the homogenate at -80 °C for up to 1 month. Cell lysates must be further diluted before assaying (1:5 or greater).

Testdurchführung:

Each triglyceride standard and sample should be assayed in duplicate or triplicate. Additionally, each sample should be tested without lipase to determine free glycerol background. A freshly prepared standard curve should be used each time the assay is performed.

1. Add 10 μL of the diluted triglyceride standards or samples to the 96-well microtiter plate.
2. Maintain all components/mixtures at 4 °C. According to Table 2 (below), prepare the desired volume of Reaction Mixture (based on the # of tests) in the following sequence: a. In a tube, add the appropriate volume of deionized water. b. To the water add the corresponding volume of 10X Assay Buffer. Mix well. c. Add the corresponding volume of 5X Enzyme Mixture. d. Next, add the corresponding volume of 10X Lipase Solution. 4 Note: To determine background free glycerol signal, this step should be skipped (without lipase). Deionized water should be used to make up the volume. e. Finally, add the corresponding volume of 200X Colorimetric Probe. Mix well and immediately use. Note: Reaction Mixture will appear slightly pink in color. This is normal background and should be subtracted from all absorbance values. Deionized 10X Assay 5X Enzyme 10X Lipase 200X Total Volume # of Tests in Water Buffer Mixture Solution Colorimetric of Reaction 96-well Plate (mL) (mL) (mL) (mL) Probe (μL) Mixture (mL) (90 μL/test) 4.950 1 2 1 50 9 100 2.475 0.5 1 0.5 25 4.5 50 0.990 0.2 0.4 0.2 10 1.8 20 Table
3. Preparation of Reaction Mixture
4. Transfer 90 μL of the above Reaction Mixture to each well (already containing 10 μL of triglyceride standard or sample).
5. Cover the plate wells to protect the reaction from light.
6. Incubate at room temperature for 30 minutes on an orbital shaker.
7. Read absorbance in the 540-570 nm range on a microplate reader.
8. Calculate the concentration of triglyceride within samples by comparing the sample absorbance to the standard curve. Negative controls (without triglyceride) should be subtracted. Absorbance from free glycerol should also be deducted for true triglyceride values.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -80 °C

Informationen zur Lagerung: Store entire kit at -80°C. Avoid multiple freeze/thaws by aliquoting. The Colorimetric Probe is light sensitive and should be maintained in amber tubes.

Referenzen

Armstrong, Adams, Zilisch, Bretl, Sato, Anderson, Zahrt: "Rv2744c Is a PspA Ortholog That Regulates Lipid Droplet Homeostasis and Nonreplicating Persistence in Mycobacterium tuberculosis." in: Journal of bacteriology, Vol. 198, Issue 11, pp. 1645-61, (2016) (PubMed).

Gulhane, Murray, Lourie, Tong, Sheng, Wang, Kang, Schreiber, Wong, Magor, Denman, Begun, Florin, Perkins, Cuív, McGuckin, Hasnain: "High Fat Diets Induce Colonic Epithelial Cell Stress and Inflammation that is Reversed by IL-22." in: Scientific reports, Vol. 6, pp. 28990, (2016) (PubMed).

Chellan, Yan, Sontag, Reardon, Hofmann Bowman: "IL-22 is induced by S100/calgranulin and impairs cholesterol efflux in macrophages by downregulating ABCG1." in: Journal of lipid research, Vol. 55, Issue 3, pp. 443-54, (2014) (PubMed).

Marino, Menghini, Fabrizi, Casagrande, Mavilio, Stoehr, Candi, Mauriello, Moreno-Navarrete, Gómez-Serrano, Peral, Melino, Lauro, Fernandez Real, Federici: "ITCH deficiency protects from diet-induced obesity." in: Diabetes, Vol. 63, Issue 2, pp. 550-61, (2014) (PubMed).

Antigendetails

Hintergrund: Triglycerides (TAG) are a type of lipid in the blood, serving as an energy source and playing a key role in metabolism. Triglycerides are the digestive end product of breaking down dietary fats. Any extra carbohydrates and fats that are not immediately used are chemically converted into triglycerides. In the intestines, secreted enzyme lipases hydrolyse the triglyceride ester bond, yielding glycerol and free fatty acids in a process called lipolysis. Enterocytes then absorb and repackage the fragments with cholesterol to form chylomicrons, a major lipoprotein transport particle. In the liver, hepatic lipases also break down triglycerides to assemble another lipoprotein particle (VLDL) from triglycerides, cholesterol, and apolipoproteins.