428901 | Cholesterol/Cholesteryl Ester Quantitation Kit

428901
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      Overview

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      Key Specifications Table

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      428901-1KIT
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          Description
          OverviewA sensitive assay for the quantitative measurement of cholesterol and/or cholesteryl ester by a colorimetric or a fluorometric method. Measurement by spectrophotometry at OD =570 nm or fluorometry at Ex/Em =535/587 nm. This assay can measure cholesterol itself, total cholesterol (cholesterol + cholesterol ester) by adding cholesterol esterase to the reaction, or only cholesteryl ester by subtracting the value of cholesterol from the total value of cholesterol plus cholesteryl esters. The fluorometric assay can detect 0.02-1 µg/well and is 4-10 fold more sensitive than colorimetric assay. Kit contains sufficient reagents for 100 tests.
          Catalogue Number428901
          Brand Family Calbiochem®
          Materials Required but Not Delivered Spectrophotometer capable of reading absorbance at 570 nm or a fluorimeter capable of measuring fluorescence at an excitation wavelength of ~535 nm and an emission wavelength of ~587 nm
          96-well plate
          Chloroform-NP-40 (1% NP-40 in pure chloroform) (for preparation of cell lysates and tissue extracts)
          References
          Product Information
          Form100 Tests
          Format96-well plate
          Kit containsCholesterol Reaction Buffer, Cholesterol Probe, Dimethylsulfoxide, Enzyme Mix, Cholesterol Esterase, Cholesterol Standard, and a user protocol.
          Applications
          Biological Information
          Assay range0.2-10 µg/well
          Sample TypeCells, tissues, serum
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Storage and Shipping Information
          Ship Code Blue Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Storage -20°C
          Storage ConditionsUpon arrival store the entire contents of the kit at -20°C.
          Protect from Light Protect from light
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsCholesterol Reaction Buffer, Cholesterol Probe, Dimethylsulfoxide, Enzyme Mix, Cholesterol Esterase, Cholesterol Standard, and a user protocol.
          Specifications

          Documentation

          SDS

          Title

          Safety Data Sheet (SDS) 

          Certificates of Analysis

          TitleLot Number
          428901
          User Protocol

          Revision27-January-2011 JSW
          Form100 Tests
          Format96-well plate
          StorageUpon arrival store the entire contents of the kit at -20°C.
          Principles of the assayThe Calbiochem® Cholesterol/Cholesteryl Ester Quantitation Kit provides a simple method for sensitive quantification of cholesterol, cholesteryl ester, or both by either colorimetric or fluorometric methods. A large portion of the cholesterol in blood is in the form of cholesteryl esters. Cholesterol esterase hydrolyzes cholesteryl ester into cholesterol. Cholesterol is then oxidized by cholesterol oxidase to yield H₂O₂. The resulting H₂O₂ interacts with a sensitive cholesterol probe to produce resorufin, which can be detected by spectrophotometrically 570 nm or by fluorometric detection at an excitation wavelength of ~535 and an emission wavelength of ~587 nm. The assay will detect cholesterol alone (without adding cholesterol esterase), total cholesterol (cholesterol + cholesteryl ester) by adding cholesterol esterase to the reaction, or cholesteryl ester alone by subtracting the value of cholesterol from the total value of cholesterol and cholesteryl esters.
          Materials provided• Cholesterol Reaction Buffer (Kit Component No. KP31682): 1 bottle, 25 ml
          • Cholesterol Probe (Kit Component No. KP31683): 1 vial, 200 µl (in DMSO, anhydrous)
          • Enzyme Mix (Kit Component No. KP31685): 1 vial, lyophilized
          • Cholesterol Esterase (Kit Component No. KP31686): 1 vial, lyophilized
          • Cholesterol Standard (Kit Component No. KP31687): 1 vial, 100 µl, supplied at 5 µg/µl
          Materials Required but not provided Spectrophotometer capable of reading absorbance at 570 nm or a fluorimeter capable of measuring fluorescence at an excitation wavelength of ~535 nm and an emission wavelength of ~587 nm
          96-well plate
          Chloroform-NP-40 (1% NP-40 in pure chloroform) (for preparation of cell lysates and tissue extracts)
          Preparation1. Serum: The recommended volume of serum is 0.5-5 µl. Serum samples should be diluted 10-fold in Cholesterol Assay Buffer.
          2. Cell Lysates and Tissue Extracts: Homogenize 1 x 106 cells or 10 mg tissue in 200 µl Chloroform:Isopropanol:NP-40 (7:11:0.1) in a microhomogenizer. Centrifuge for 5-10 min at 14,000 rpm in a microcentrifuge. Avoiding the whitish solid protein layer, collect the organic phase (lower phase) and transfer it to a clean tube; air-dry at 50°C, and vacuum-dry for 30 min to remove residual chloroform. Dissolve the dried lipids in 200 µl Cholesterol Reaction Buffer by vortexing extensively for 5 min. Use 1-50 µl extracted sample per assay. 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 unkown samples, we suggest testing differant amounts of sample to ensure that the readings are within the linear portion of the standard curve.
          Reagent preparationNote: Allow reagents to warm to room temperature. Briefly centrifuge all vials before opening.
          • Cholesterol Probe: Ready to use as supplied. Warm to room temperature to thaw the DMSO solution before use. Aliquot and freeze at -20°C; protect from light. Cholesterol Probe is stable for up to 2 months at -20°C.
          • Cholesterol Esterase: Dissolve the Cholesterol Esterase in 220 µl Cholesterol Reaction Buffer prior to use. Aliquot and freeze at -20°C. Reconstituted Cholesterol Esterase is stable for up to 2 months at -20°C.
          • Enzyme Mix: Dissolve the Enzyme Mix in 220 µl Cholesterol Reaction Buffer prior to use. Aliquot and freeze at -20°C. Reconstituted Enzyme Mix is stable for up to 2 months at -20°C.
          • Standard Curve, Colorimetric Detection: Dilute the Cholesterol Standard to a concentration of 0.5 µg/µl by adding 20 µl Cholesterol Standard to 180 µl Cholesterol Reaction Buffer; mix well. Add the indicated volumes of diluted Cholesterol Standard and Cholesterol Reaction Buffer (see table below) to individual designated standard wells.

          Table 1: Sample Dilutions


          • Standard Curve, Fluorometric Detection: Dilute the Cholesterol Standard to 50 ng/µl by adding 10 µl Cholesterol Standard to 990 µl Cholesterol Reaction Buffer; mix well. Add the indicated volumes of diluted Cholesterol Standard and Cholesterol Reaction Buffer (see table below) to individual designated standard wells.

          Table 2: Sample Dilutions


          Note: For the standard curve (colorimetric and fluorescent detection), cholesterol esterase MUST be added to the reaction mix for detecting either total cholesterol or free cholesterol.
          • Reaction Mix: Prepare the following Reaction Mix; prepare only enough reaction mix for the number of assays to be performed. The total volume of reaction mix in each well is 50 µl.

          44 µl Cholesterol Reaction Buffer
          2 µl Cholesterol Probe
          2 µl Enzyme Mix
          2 µl Cholesterol Esterase*

          *Note: Cholesterol Esterase hydrolyzes cholesteryl ester into cholesterol. If you want to detect cholesterol only, omit the Cholesterol Esterase. With the addition of Cholesterol Esterase, the assay detects both cholesterol and cholesteryl esters. If you want to detect cholesteryl esters only, subtract the value of cholesterol from the total value of both cholesterol and cholesteryl esters.
          Detailed protocol1. Add samples to designated wells.
          2. Add 50 µl Reaction Mix to each well and incubate for 1 h at 37°C. Protect the plate from light during the incubation.
          3. Measure the absorbance at 570 nm for colorimetric detection or at an excitation wavelength of ~535 nm and emission wavelength of ~ 590 nm for fluorescent detection.
          4. Subtract the value of the no-cholesterol control from all samples (Note: this background reading can be significant and must be subtracted from sample readings). Plot the concentration of the Cholesterol Standards against the absorbance or fluorescence (see example below). Apply sample readings to the standard curve. Cholesterol concentration in samples can be calculated as follows:

          C = A/V (µg/micro;l)

          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 1 µg/µl = mg/dL.
          Assay characteristics and examples

          Figure 1: Detection of Cholesterol/Cholesteryl Ester.

          Cholesterol/Cholesteryl Ester was quantitated using the Cholesterol Probe by colorimetric (A) and fluorescent (B) detection according to the Detailed Protocol outlined above. Background from the no-cholesterol control has been subtracted from each value.





          Limitations of the assayFluorometric detection is ~10-fold more sensitive than colorimetric detection. Using fluorometric detection, the assay can detect 0.2-1 µg/well.
          Assay Range0.2-10 µg/well
          Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals, Inc.