428900 | LCAT Activity Assay Kit, Fluorometric

428900
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      Overview

      Key Spec Table

      Detection Methods
      Fluorometric

      Pricing & Availability

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      428900-1KIT
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      Discontinued
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          Description
          Overview Assay kit useful for the quantitative measurement of lecithin:cholesterol acyltransferase activity (LCAT) in human plasma. LCAT mediates the formation of cholesteryl esters in human plasma by transferring an acyl chain from the sn-2 position of phosphatidylcholine to cholesterol.
          Catalogue Number 428900
          Brand Family Calbiochem®
          Synonyms Lecithin:Cholesterol Acyltransferase Activity Assay Kit, Fluorometric
          Application Data
          The non-hydrolyzed LCAT Substrate fluoresces at 470 nm. Upon hydrolysis by LCAT, a monomer is released from the LCAT Substrate that fluoresces at 390 nm. LCAT activity is assessed as a change in 470/390 emission intensity.

          Normal human plasma is assayed according to the Detailed Protocol above. Over time the 470/390 ration decreases, indicating LCAT activity in the sample.
          References
          Product Information
          Detection method Fluorometric
          Form 100 Tests
          Kit contains LCAT Substrate, READ Reagent, and a user protocol.
          Applications
          Biological Information
          Assay time 4.5 - 8.5 h
          Sample Type Plasma
          Species human
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          R Phrase R: 36/38

          Irritating to eyes and skin.
          S Phrase S: 26

          In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
          Product Usage Statements
          Intended use The Calbiochem® LCAT Activity Assay Kit, Fluorometric is intended for the quantitative measurement of lecithin:cholesterol acyltransferase activity (LCAT) in normal human plasma. This kit allows the quantitation of fold of increase or decrease of enzymatic activity among samples, this kit also allows the quantitation of the % change of 470/390 ratio with time, which can be used to define LCAT enzymatic activity. This kit does not allow the measurement of substrate concentration or the amount of hydrolyzed substrate.
          Storage and Shipping Information
          Ship Code Blue Ice Only
          Toxicity Irritant
          Storage Multiple storage requirements
          Storage Conditions Upon arrival store the LCAT Substrate at -20°C and the READ Reagent at 4°C.
          Protect from Light Protect from light
          Packaging Information
          Transport Information
          Supplemental Information
          Kit contains LCAT Substrate, READ Reagent, and a user protocol.
          Specifications

          Documentation

          MSDS

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          Certificates of Analysis

          TitleLot Number
          428900
          User Protocol

          Revision 13-July-2012 JSW
          Synonyms Lecithin:Cholesterol Acyltransferase Activity Assay Kit, Fluorometric
          Form 100 Tests
          Detection method Fluorometric
          Species human
          Storage Upon arrival store the LCAT Substrate at -20°C and the READ Reagent at 4°C.
          Intended use The Calbiochem® LCAT Activity Assay Kit, Fluorometric is intended for the quantitative measurement of lecithin:cholesterol acyltransferase activity (LCAT) in normal human plasma. This kit allows the quantitation of fold of increase or decrease of enzymatic activity among samples, this kit also allows the quantitation of the % change of 470/390 ratio with time, which can be used to define LCAT enzymatic activity. This kit does not allow the measurement of substrate concentration or the amount of hydrolyzed substrate.
          Principles of the assay LCAT mediates the formation of cholesteryl esters in human plasma by transferring an acyl chain from the sn-2 position of phosphatidylcholine to cholesterol. Using the LCAT Activity Assay, Fluorometric, human plasma samples are incubated with LCAT Substrate, a fluorescent substrate that emits fluorescence at 470 nm. When the substrate is hydrolyzed by LCAT, a monomer is released that emits fluorescence at 390 nm. The LCAT activity is assessed as a change in 470/390 emission intensity.










          Figure 1: Assay Principle

          The non-hydrolyzed LCAT Substrate fluoresces at 470 nm. Upon hydrolysis by LCAT, a monomer is released from the LCAT Substrate that fluoresces at 390 nm. LCAT activity is assessed as a change in 470/390 emission intensity.

          Materials provided There are sufficient reagents for 100 tests.

          •LCAT Substrate (Kit Component No. KP23001): 1 vial, 100 µl
          •READ Reagent (Kit Component No. KP23002): 1 vial, 350 µl
          Materials Required but not provided •LCAT Assay Buffer (150 mM NaCl, 10 mM Tris-HCl, 4 mM 2-mercaptoethanol, 1 mM EDTA, 7.4)
          •READ Reagent Reconstitution Buffer (150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4)
          Reagent preparation •READ Reagent: reconstitute the vial of READ Reagent with 29.7 ml READ Reagent Reconstitution Buffer.
          Detailed protocol 1. Mix 1 µl LCAT Substrate 200 µl LCAT Assay Buffer with sample containing LCAT (e.g., 3-5 µl normal human plasma) and incubate for 4-8 h at 37°C.
          2. Add 100 µl of the mixture in step 1 to 300 µl READ Reagent and vortex. Read the fluorescence at an excitation wavelength of 340 nm and an emission wavelength of 390 nm and 470 nm. DO NOT incubate the samples in the presence of READ Reagent, as this will inactivate the LCAT in the sample.

          NOTE: The emission intensities at 390 nm and 470 nm represent the emission of the substrate hydrolyzed (390 nm) and not hydrolyzed (470 nm). In the presence of LCAT, a ratio of the two emission intensities (470/390) will indicate an increase in concentration of the monomer, which emits at 390 nm, and the simultaneous decrease in concentration of the non-hydrolyzed LCAT Substrate, which emits at 470 nm.
          Example data

          Figure 2: Example Data

          Normal human plasma is assayed according to the Detailed Protocol above. Over time the 470/390 ration decreases, indicating LCAT activity in the sample.

          Registered Trademarks Calbiochem® is a registered trademark of Merck KGaA, Darmstadt.
          Interactive Pathways™ is a trademark of Merck KGaA, Darmstadt.