Triglyceride Quantification Kit

Antigen: Triglyceride

Applikation: Biochemisches Assay (BCA)

Produktnummer: ABIN411709

Produktbeschreibung

Beschreibung: Triglycerides (TG) are the main constituent of vegetable oil, animal fat, LDL and VLDL, and play an important role as transporters of fatty acids as well as serving as an energy source. TG are broken down into fatty acids and glycerol, after which both can serve as substrates for energy producing and metabolic pathways. High blood levels of TG are implicated in atherosclerosis, heart disease and stroke as well as in pancreatitis. The Triglyceride Quantification Kit provides a sensitive, easy assay to measure TG concentration in variety of samples. In the assay, TG are converted to free fatty acids and glycerol. The glycerol is then oxidized to generate a product which reacts with the probe to generate colorimetric (spectrophotometry at lambda = 570 nm) and fluorometric (Ex/Em = 535/587 nm) methods. The kit can detect 2 pmol-10 nmol (or 2~10000 µM range) of triglyceride in various samples.

Anwendungen

Protokoll: 1. Standard Curve Preparation: For the colorimetric assay, Dilute 40 µl of the 1 mM Triglyceride into 160 µl Assay Buffer, mix to generate 0.2 mM Triglyceride standard. Add 0, 10, 20, 30, 40, 50 µ l of the 0.2 mM Triglyceride Standard into wells individually. Adjust volume to 50 µ l/well with Triglyceride Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of Triglyceride Standard. For the fluorometric assay, dilute the Triglyceride Standard to 0.01- 0.1 mM with the Triglyceride Assay Buffer (Detection sensitivity is 10-100 fold higher for a fluorometric than a colorimetric assay). Follow the procedure as the colorimetric assay. 2. Sample Preparation:* Prepare test samples to a final volume of 50 µ l/well with Triglyceride Assay Buffer in a 96-well plate. We suggest testing several dilutions of your sample to make sure the readings are within the standard curve range. *Notes: Serum contains 0.1-6 mM triglyceride, which can be tested directly. For tissues (100 mg) or cells (10 millions) or other non-aqueous samples, homogenize samples in 1 ml solution containing 5% Triton-X100 in water, slowly heat the samples to 80-100 ° C in a water bath for 2-5 minutes or until the Triton X-100 becomes cloudy, then slowly cool down to room temperature. Repeat the heating one more time to solublize all triglyceride into solution. Centrifuge for 5 min (top speed using a microcentrifuge) to remove any insoluble materials. Dilute 10 folds with dH 2 O before the assay. 3 Lipase: Add 2 µ l of lipase to each standard and sample well. Mix and incubate 20 min at room temperature to convert triglyceride to glycerol and fatty acid. Note: If samples contain glycerol, do a sample background control, omit the lipase addition, so that the assay measure glycerol background only, not triglyceride. 4. Triglyceride Reaction Mix: Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 µ l Reaction Mix: 46 µ l Triglyceride Assay Buffer 2 µ l Triglyceride Probe* 2 µ l Triglyceride Enzyme Mix* *

Applikationshinweise: Note: If samples contain glycerol, do a sample background control, omit the lipase addition, so that the assay measure glycerol background only, not triglyceride. 4. Triglyceride Reaction Mix: Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 µ l Reaction Mix: 46 µ l Triglyceride Assay Buffer 2 µ l Triglyceride Probe* 2 µ l Triglyceride Enzyme Mix* *For the fluorometric assay, you may use 10% of the Probe to decrease the background readings, therefore increase detection sensitivity. 5. Mix well. Add 50 µ l of the Reaction Mix to each well containing the Triglyceride Standard, test samples and controls. Mix well. Incubate at room temperature for 30-60 minutes-60 minutes gives slightly better result, protect from light. 6. Measure O.D. 570 nm for colorimetric assay or Ex/Em = 535/590 nm for fluorometric assay in a microtiter plate reader. The reaction is stable for at least two hours. 7. Calculations: Correct background by subtracting the value derived from the 0 triglyceride standard from all sample readings. Plot the standard curve. Apply sample readings to the standard curve. Triglyceride concentration can then be calculated: C = Ts / Sv nmol/ µ l or µ mol/ml or mM Where: Ts is triglyceride amount from standard curve (nmol). Sv is the sample volume (before dilution) added in sample wells ( µ l). VI.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Publikationen

Publikationen: Fierz, Novosyadlyy, Vijayakumar et al.: "Mammalian target of rapamycin inhibition abrogates insulin-mediated mammary tumor progression in type 2 diabetes." in: Endocrine-related cancer, Vol. 17, Issue 4, pp. 941-51, 2010 (PubMed).

Yu, Wu, Cheng et al.: "The function of porcine PPARγ and dietary fish oil effect on the expression of lipid and glucose metabolism related genes." in: The Journal of nutritional biochemistry, Vol. 22, Issue 2, pp. 179-86, 2011 (PubMed).

Segatto, Trapani, Marino et al.: "Age- and sex-related differences in extrahepatic low density lipoprotein receptor." in: Journal of cellular physiology, 2010 (PubMed).

Kawaratani, Tsujimoto, Kitazawa et al.: "Therapeutic effects of cytokine modulator Y-40138 in the rat alcoholic liver disease model." in: Journal of gastroenterology and hepatology, 2011 (PubMed).

Tsurutani, Fujimoto, Takemoto et al.: "The roles of transforming growth factor-β and Smad3 signaling in adipocyte differentiation and obesity." in: Biochemical and biophysical research communications, Vol. 407, Issue 1, pp. 68-73, 2011 (PubMed).

Chung, Kuo, Chen et al.: "YC-1 rescues cancer cachexia by affecting lipolysis and adipogenesis." in: International journal of cancer. Journal international du cancer, 2011 (PubMed).

Ellefson, Lakner, Hamilton et al.: "Neonatal androgenization exacerbates alcohol-induced liver injury in adult rats, an effect abrogated by estrogen." in: PLoS ONE, Vol. 6, Issue 12, pp. e29463, 2011 (PubMed).