Cholesterol Quantitation Kit

Antigen: Cholesterol (CH)

Applikation: Biochemisches Assay (BCA)

Produktnummer: ABIN411647

Produktbeschreibung

Weitere Bezeichnung: Cholesterol

Beschreibung: The Cholesterol/Cholesteryl Ester Quantitation Kit provides a simple method for sensitive quantification of free cholesterol, cholesteryl esters, or both by colorimetric or fluorometric methods. Majority of the cholesterol in blood is in the form of cholesteryl esters which can be hydrolyzed to cholesterol by cholesterol esterase. Cholesterol is then oxidized by cholesterol oxidase to yield H 2 O 2 which reacts with a sensitive cholesterol probe to produce color (lambda max = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The assay detects total cholesterol (cholesterol and cholesteryl esters) in the presence of cholesterol esterase, or free cholesterol in the absence of cholesterol esterase in the reaction. Cholesteryl ester can be determined by subtracting the value of free cholesterol from the total (cholesterol plus cholesteryl esters). Contents: III. Storage and Handling: Store kit at -20°C, protect from light. Allow Assay Buffer and Cholesterol Probe to warm to room temperature before use. Keep enzymes and cholesterol standard on ice while using. IV. Reagent Preparation: Cholesterol Probe: Ready to use as supplied. Warm to room temperature to thaw the DMSO solution. Store at -20°C, protect from light. Use within two months. Cholesterol Esterase: Dissolve in 220 µl Cholesterol Assay Buffer before use. Aliquot and store at -20°C. Use within two months. Enzyme Mix: Dissolve in 220 µl Cholesterol Assay Buffer before use. Aliquot and store at -20°C. Use within two months. V. Cholesterol Assay Protocol: 1. Standard Curve Preparations: For the colorimetric assay, dilute the Cholesterol Standard to 0.25 µg/µl by adding 20 µl of the Cholesterol Standard to 140 µl of Cholesterol Assay Buffer, mix well. Add 0, 4, 8, 12, 16, 20 µl into a series of wells. Adjust volume to 50 µl/well with Cholesterol Assay Buffer to generate 0, 1, 2, 3, 4, 5 µg/well of the Cholesterol Standard. For the fluorometric assay, dilute the Cholesterol Standard to 25 ng/µl by adding 10 µl of the Cholesterol Standard to 790 µl of Cholesterol Assay Buffer, mix well. Follow the same protocol above to generate 0, 0.1, 0.2, 0.3, 0.4, 0.5 µg/well of the Cholesterol Standard. 2. Sample Prep: Serum samples (0.5-5 µl/assay) should be diluted 10-fold in the Cholesterol Assay Buffer. For cells or tissue samples, 10 6 cells or 10 mg tissue can be extracted with 200 µl of chloroform : Isopropanol : NP-40 (7:11:0.1) in a micro-homogenizer. Spin the extract 5-10 minutes at 15,000 xg in a centrifuge. Transfer all of the liquid (organic phase) avoiding the pellet, to a new tube, air dry at 50°C to remove chloroform. Put the samples under vacuum for 30 min to remove trace organic solvent. Dissolve dried lipids with 200 µl of Cholesterol Assay Buffer by sonicating or vortexing until homogeneous (it is OK if the solution becomes cloudy). The extraction procedure can be scaled up if larger amounts of sample are desired. Use 1- 50 µl of extracted sample per assay. Then adjust the volume to 50 µl/well with Cholesterol Assay Buffer. For unknown samples, we suggest testing different amounts of samples to ensure that readings are within the linear portion of the standard curve. 3. Reaction Mix Preparation: Mix enough reagent for the number of assays performed: For each well, prepare a total 50 µl Reaction Mix containing: 44 µl Cholesterol Assay Buffer 2 µl Cholesterol Probe 1 2 µl Cholesterol Enzyme Mix 2 µl Cholesterol Esterase 2.3 Notes: 1. For the fluorometric assay, use 0.4 µl of the probe for each reaction to decrease fluorescence background. The fluorometric assay is over 10 fold more sensitive than the colorimetric assay. 2. Cholesterol Esterase hydrolyzes cholesteryl ester to cholesterol. If you want to detect free cholesterol only, omit the Cholesterol Esterase in the reaction. In the presence of Cholesterol Esterase, the assay detects both free cholesterol and cholesteryl esters. If you want to determine Cholesteryl Ester only, subtract the value of free cholesterol from the value of total cholesterol (Cholesterol and Cholesteryl Ester). 3. The Cholesterol Standard contains a mixture of free cholesterol and cholesterol esters in a similar ration of serum. Cholesterol Esterase must be added to the standard curve reaction to convert all cholesterol. 4. Add 50 µl of the Reaction Mix to each well containing standard or test samples. 5. Incubate the reaction for 60 minutes at 37°C, protect from light. 6. Measure absorbance at 570nm for the colorimetric assay or fluorescence at Ex/Em = 535/590 nm in a microplate reader. 7. Calculations: Subtract the 0 standard background reading from all readings. Plot the standard curve. Apply sample readings to the standard curve. Cholesterol concentration in samples can be calculated: C = A/V (µg/µl) Where: A = amount of cholesterol determined from Standard Curve (in µg). V= volume of sample added into the reaction well (in µl). Cholesterol molecular weight: 386.65. 1µg/µl = 100 mg/dL. Colorimetric Assay Fluorometric Assay Cholesterol/Cholesteryl Ester was quantified using colorimetric (A) and fluorometric (B) methods according to the kit instructions. Background from the 0 standard reading (without cholesterol) has been subtracted from all readings. The fluorometric assay is over ten-fold more sensitive than the colorimetric assay. Component Volume Cap Code Part No. Cholesterol Assay Buffer 25 ml WM K603-100-1 Cholesterol Probe (in DMSO, anhydrous) 200 µl Red K603-100-2A Enzyme Mix (lyophilized) 1 vial Green K603-100-4 Cholesterol Esterase (lyophilized) 1 vial Blue K603-100-5 Cholesterol Standard (2 µg/µl) 100 µl Yellow K603-100-6 y = 89532x + 739.1 0 10 20 30 40 50 0 0.1 0.2 0.3 0.4 0.5 RFU Cholesterol (µg/well) y = 0.2501x - 0.0061 0 0.4 0.8 1.2 0 1 2 3 4 5 Absorbance 570n m Cholesterol (µg/well)

Anwendungen

Beschränkungen: Nur für Forschungszwecke einsetzbar

Publikationen

Publikationen: Mankidy, Ahiahonu, Ma et al.: "Membrane plasmalogen composition and cellular cholesterol regulation: a structure activity study." in: Lipids in health and disease, Vol. 9, pp. 62, 2010 (PubMed).

Zhang, Thomas, Marshall et al.: "Nicotinic acetylcholine receptor alpha1 promotes calpain-1 activation and macrophage inflammation in hypercholesterolemic nephropathy." in: Laboratory investigation; a journal of technical methods and pathology, 2010 (PubMed).

Zhang, Marshall, Thomas et al.: "In vivo knockdown of nicotinic acetylcholine receptor α1 diminishes aortic atherosclerosis." in: Atherosclerosis, Vol. 215, Issue 1, pp. 34-42, 2011 (PubMed).

Asada, Kuroda, Aoyagi et al.: "Disturbed apolipoprotein A-I-containing lipoproteins in fish-eye disease are improved by the lecithin:cholesterol acyltransferase produced by gene-transduced adipocytes in vitro." in: Molecular genetics and metabolism, Vol. 102, Issue 2, pp. 229-31, 2011 (PubMed).

Postigo, Genre, Iglesias et al.: "Exacerbation of collagen type II-induced arthritis in ApoE deficient mice in association with the expansion of Th1 and Th17 cells." in: Arthritis and rheumatism, 2011 (PubMed).

Kim, Ee, Jittiwat et al.: "Increased expression of acyl-coenzyme A: cholesterol acyltransferase-1 and elevated cholesteryl esters in the hippocampus after excitotoxic injury." in: Neuroscience, Vol. 185, pp. 125-34, 2011 (PubMed).