MTP Activity Assay Kit Protocol

Supplied by Roar Biomedical, Inc

Catalog Number: MAK110
Storage Temperature 2-8 °C

Components

The kit is sufficient for 100 assays in 200 µL total assay volume.

Donor Particle                                0.4 mL
    Catalog Number MAK110A

Acceptor Particle                            0.4 mL
    Catalog Number MAK110B

MTP Assay Buffer                           20 mL
    Catalog Number MAK110C

Reagents Required but Not Provided

  • Reagents and Equipment Required but Not Provided.
  • 96 well U-bottom black plates for fluorescence assays, with adhesive sealers
  • 37 °C water bath incubator
  • Fluorescence multiwell plate reader
  • Spectrally pure Isopropanol (Catalog Number 34863)
  • PMSF (Catalog Number P7626), for homogenization of HepG2 cells
  • Leupeptin (Catalog Number L2884), for homogenization of HepG2 cells
  • CP-346086 (Catalog Number PZ0103), 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 2–8 °C, protected from light, is recommended. DO NOT FREEZE.

Components are stable for 1 year, if stored properly.

Procedure

All samples and standards should be run in duplicate. 

Standards for Fluorometric Detection

  1. Prepare six test tubes labeled from T0 to T5 each containing 1 mL isopropanoland add an additional 1 mL of isopropanol to T5.
     
  2. Pipette 5 µL of Donor Particle into test tube T5 and thoroughly mix (vortex) to adequately disperse the donor particle in the isopropanol.
    Note: The concentration of fluorescent substrate in the donor particle is listed on the vial label.
     
  3. Make four serial 2-fold dilutions (transfer 1 mL of previous standard solution to a tube with 1 mL of 2-propanol). For example transfer 1 mL of the mixture in tube T5 to tube T4 and vortex. Use tube T0 with isopropanol only as the 0 (Blank) Standard.
    Note: DO NOT incubate the standards.
     
  4. Measure the fluorescence intensity (λex = 465/λem = 535) of the standards from tubes T0 to T5. For example, pipette 100 µL of each tube to a separate well of a plate and read the plate.
     
  5. Create a standard curve by plotting the fluorescence intensity units of each standard versus the pmole amounts of Donor Particle in each standard. Plot a standard curve (FIU to nmoles of label) and determine the slope (m) and intercept (b). This is the standard curve for calculating nmoles of label from fluorescence intensity units.

Sample Preparation – Homogenization of HepG2 Cells

  1. Grow HepG2 cells in 75 cm2 T-flasks until confluent.
     
  2. Prepare a sufficient amount of Homogenization Buffer (10 mM Tris, pH 7.4, with 150 mM NaCl and 1 mM EDTA).
     
  3. To 100 mL of Homogenization Buffer, add 0.5 mL of 100 mM PMSF (Catalog Number P7626) in ethanol and 2 mL of 1 mg/mL leupeptin (Catalog Number L2884) in Tris-saline.
     
  4. Suspend the cells from 6 flasks in a total of 5 mL of homogenization buffer (protein concentration ~10 mg/ml).
     
  5. Sonicate the suspension on ice with five 5-second bursts in a 550 W sonicator (power setting: 4) fitted with a microtip.
     
  6. Use 100 µg of protein homogenate in the MTP activity assay.
    Note: It is not necessary to spin down cell debris to make a low-speed supernatant, nor is it necessary to partially purify membranes to assay MTP activity.

Assay Reaction

  1. Set up the Master Reaction Mix according to the scheme in Table 1. Add 190 µL of the Master Reaction Mix to each reaction (well).

    Table 1.
    Master Reaction Mix

    Reagent Volume
    MTP Assay Buffer 182 µL
    Donor Particle 4 µL
    Acceptor Particle 4 µL
     
  2. Add 10 µL of desired MTP source (homogenized HepG2 cells or partially purified MTP) to the appropriate wells. For a sample blank, add 10 µL of Assay buffer in place of MTP source.
    Notes: For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.
     
    Sample volumes may need to be adjusted depending on the specific activity of the MTP source in the assay. For samples with low activity, decrease the buffer volume and increase the sample volume in the assay. Alternatively, microplates containing samples may be incubated overnight at 25 °C. MTP remains active at 25 °C.
     
  3. Seal plate and incubate for 3–6 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.
     
  4. Measure the increase in fluorescence of samples using a fluorometer (λex = 465/λem = 535 nm). Determine the fluorescence intensity in the plasma or serum samples by subtracting the fluorescence intensity of the sample blank from each sample.
     
  5. Calculate pmoles of label in the assay by subtracting the intercept (b) from (y) and then dividing by the slope (m) of the standard curve.

Assay Validation Procedure
Purified MTP must be used with CP-346086. In this procedure, MTP was purified from rat liver microsomes.

Reagent Preparation

  • Buffer A – 50 mL of 100 mM Tris, pH 7.4, with 100 mM KCl and 10 mM MgCl2
  • 50 mM Tris, pH 7.4, with 0.54% deoxycholate
  • Buffer B – 100 mM Tris, pH 7.4, with 1.5 M NaCl and 10 mM EDTA
  • 1% BSA (not fatty acid free)

Sample Preparation
Note:
Chill buffers and rotor

  1. Thaw rat liver microsomes (0.5 mL, total protein = 20 mg/mL) and dilute to 3.5 mg/mL with Buffer A so that the resulting microsome solution is 50 mM Tris, pH 7.4, with 50 mM KCl and 5 mM MgCl2 at 3.5 mg/mL total protein. For example, take 1.429 mL of Buffer A and mix with 0.929 mL of water and 0.5 mL microsomes.
     
  2. Add 0.1x volume of deoxycholate solution while vortexing and keep on ice. For example, add three separate 95 µL aliquots while vortexing and chilling.
     
  3. Incubate on ice for 30 minutes.
     
  4. Centrifuge at 105,000 x g for 75 minutes. Recover the supernatant while discarding the pellet.
     
  5. Add 0.1x volumes of Buffer B. Use this directly in the validation assay with CP-346086.

Validation Reaction

  1. Dissolve CP-346086 in DMSO at the appropriate concentrations (for example, 5.5, 1.11, 0.55, 0.111, and 0.011 µM). 
     
  2. Set up the Validation Master Reaction Mix according to the scheme in Table 2. Add 94 µL of the Validation Master Reaction Mix to for each reaction (well).
     
    Table 2.
    Validation Master Reaction Mix

    Reagent
    Volume
    MTP Assay Buffer
    80 µL
    Doner Particle
    2 µL
    Acceptor Particle
    2 µL
    1% BSA
    10 µL
     
  3. Add 1 µL of each CP-346086 dilution to separate, mixing well by pipetting. Add 5 µL of the purified MTP, again mixing well by pipetting. Generating final inhibitor concentrations of 55, 11.1, 5.5, 1.11, and 0.111 nM.
     
  4. To a Sample Blank, add 5 µL of MTP Assay Buffer and 1 µL of DMSO, in place of sample and
    CP-346086 solutions.
     
  5. Seal plate and incubate for 60 minutes at 37 °C 
     
  6. Measure the fluorescence of validation samples using a fluorometer (λex = 465/λem = 535 nm).

Figure 1.
MTP Activity in Rat Liver Microsomes

MTP Activity in Rat Liver Microsomes

 

Figure 2.
MTP Inhibition with CP-346086

MTP Inhibition with CP-346086

Materials

     
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