LCAT Activity Assay Kit

Supplied by Roar Biomedical, Inc

Catalog Number: MAK107
Storage Temperature –20 °C

The emission spectrum of the Substrate Reagent has two distinct peaks, 390 nm and 470 nm. The relative intensity of the peaks depends upon the concentration of hydrolyzed and intact substrate present in the assay. If the substrate is intact, the flourophors are in close proximity and some energy of the excited states is dissipated by radiationless transitions. The emission intensity is predominately at the less energetic 470 nm peak. After hydrolysis of the substrate by LCAT, the fluorophors are not able to energetically interact and a shift in intensity is seen in the emission spectrum as an increase in 390 nm emission with a decrease of the 470 nm emission peak. It is important to measure both 390 and 470 nm emission because the fluorescence of the substrate is affected by several assay variables.

Components

The kit is sufficient for 100 assays in 100 µL total assay volume.

Substrate Reagent                                0.1 mL
    Catalog Number MAK107A

Read Reagent                                      30 mL
    Catalog Number MAK107B

LCAT Assay Buffer                                20 mL
    Catalog Number MAK110C

Reagents and Equipment Required but Not Provided

  • 96 well polypropylene plates for assay set up
  • 96 well U-bottom black plates for fluorescence assays
  • 37 °C water bath incubator
  • Fluorescence multiwell plate reader
  • Iodoacetate (Catalog Number 57858) for assay validation

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

Briefly centrifuge vials before opening.

Storage/Stability

The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended. Components are stable for 1 year, if stored properly.

Procedure

All samples and standards should be run in duplicate. 

Single Tube Method

  1. Mix 1 µL of LCAT Substrate Reagent with 200 µL of Assay Buffer and LCAT source (3–5 µL of plasma or serum).
     
  2. Incubate for 4–8 hours at 37 °C.

  3. Add 100 µL of the incubated mixture to 300 µL of Read Reagent and then vortex. Measure the fluorescent label (λex = 340/λem = 390 and 470 nm).
     
    Note: Do not incubate the assay with Read Reagent - it will inactivate LCAT.
     

Microplate Method

  1. Combine 4 µL of sample (plasma) and 0.5 µl of Substrate Reagent into well of polypropylene reaction plate. Bring to final volume of 100 µl with LCAT Assay buffer.
     
  2. Incubate for 2.5 hours at 37 °C.
    Note: The microplate incubator must be able to rapidly raise the assay temperature to 37 °C. Large, humidified air incubators may cause problems by slowly increasing the temperature from 25 °C to only 34 °C after three hours. Floating the plate in a water bath is recommended, rather than using an air incubator.
     
  3. Add 200 µL of Read reagent to wells of polypropylene plate, mixing well by pipetting.
     
  4. Transfer 200 µL of the reaction mixture from the polypropylene plate to a black fluorescence microplate.
     
  5. Measure the increase in fluorescence of samples using a fluorometer (λex = 340/λem = 390 and 470 nm).
     
  6. Determine the ratio (λem 470/λem 390) to compare plasma LCAT activity among samples.
    Note: The fluorescence of the substrate is affected by several assay variables, such as viscosity and oxygen quenching as well as matrix effects. Better results are achieved with a ratio of the two emission intensities. Color quenching from compounds introduced into the assay or using a hemolyzed sample is also eliminated when a ratio is used.

    Figure 1.
    Iodoacetate titration into LCAT Activity Assay

MAK107-figure1

Materials

     
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