ab65387
PLTP Inhibitor Drug
Screening Kit
Instructions for Use
For the rapid, sensitive and accurate detection of
PLTP inhibition by various compounds
This product is for research use only and is not
intended for diagnostic use.
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Table of Contents
1.
Overview
3
2.
Protocol Summary
3
3.
Components and Storage
4
4.
Assay Protocol
5
5.
Data Analysis
7
6.
Troubleshooting
8
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1. Overview
Plasma phospholipid transfer protein (PLTP) is thought to play a
major role in the facilitated transfer of phospholipids between
lipoproteins and in the modulation of high-density lipoprotein (HDL)
particle size and composition. PLTP-facilitated lipid transfer activity is
related to HDL and LDL metabolism, as well as lipoprotein lipase
activity, adiposity, and insulin resistance.
Abcam’s Drug Screening Kit utilizes Rabbit serum as PLTP activity
source to screen PLTP inhibitors. The assay uses a Donor Molecule
containing
a
fluorescent
self-quenched
phospholipid
that
is
transferred to an acceptor molecule in the presence of PLTP.
PLTP-mediated transfer of the fluorescent phospholipid to the
acceptor
molecule
results
in
an
increase
in
fluorescence
(Ex/ Em = 465/ 535 nm). Inhibitor of PLTP will inhibit the lipid
transfer and therefore decrease fluorescence intensity. The kit
provides sufficient reagents for 100 PLTP inhibitor screening assays.
2. Protocol Summary
Standard Curve Preparation
Sample Preparation
Prepare and Add Reaction Mix
Measure Fluorescence
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3. Components and Storage
A. Kit Components
Item
Quantity
PLTP Donor Molecule
1 mL
PLTP Acceptor Molecule
1 mL
PLTP Assay Buffer (10X)
5 mL
Positive Control (Rabbit Serum)
300 µL
* Store kit at +20°C
B. Additional Materials Required
•
Microcentrifuge
•
Pipettes and pipette tips
•
Fluorescent microplate reader
•
96-well plate
•
Isopropanol
•
Orbital shaker
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4. Assay Protocol
Note: We recommend using a microtiter plate for the assay.
The microtiter plates should be sealed as tightly as possible
with plate sealer and incubated in a sealed, humidified chamber
to prevent evaporation.
If using a regular fluorometer for sample reading, the samples should
be diluted to 500 µl with 1X PLTP Assay Buffer before reading.
1. Standard Curve Preparation:
A standard curve is prepared by making serial dilutions of the donor
molecule in isopropanol (not included) and subsequently recording
the fluorescence intensity of each dilution, using isopropanol alone
as a blank.
a) Prepare 6 test tubes labeled T0 to T5, each contains 0.2 ml of
isopropanol; the tube labeled T5 should contain an additional
0.2 ml of isopropanol.
b) Add 2 µl Donor Molecule to T5, vortex to mix well.
c) Transfer 0.2 ml from T5 to T4. Mix and then transfer 0.2 ml from
T4 to T3. Mix and then transfer 0.2 ml from T3 to T2. Mix and
then transfer 0.2 ml from T2 to T1. The Donor Molecule solution
contains 0.1 mM labeled lipids and thus the standard curve
samples contain 0, 6.25, 12.5, 25, 50, 100 pmol Donor Molecule.
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d) Read the fluorescence intensity (Ex/Em: 465/535) of the standard
samples from T0 to T5.
e) Apply the fluorescence intensity values of the standard curve
directly to your results to express specific activity of the plasma
sample (moles/µl plasma/hr).
2. Sample Preparation:
Prepare each testing sample in 160 µl of dH2O. Add 3 µl of Rabbit
Serum.
Prepare a blank that contains no Rabbit serum as background.
Prepare a positive control assay containing 3 µl of Rabbit Serum,
but no testing inhibitors.
3. Reaction Mix: Prepare a Master Mix for each assay containing
the following:
Donor Molecule
10 µl
Acceptor Molecule
10 µl
10X PLTP Assay Buffer
20 µl
Mix well. Add 40 µl of the Master Mix into each sample including the
blank and positive control also as prepared in Step 2. Mix well and
incubate at 37°C for 30-60 min.
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5. Measurement:
Measure the fluorescence intensity of the blank, test samples, and
positive control using a fluorescence plate reader or fluorometer
(Ex/Em: 465/535).
Note:
Due to the nature of the self-quenched probe, background
fluorescence is usually high and therefore fluorescence intensity
from each sample should be corrected by subtracting the
background fluorescence intensity (without Rabbit serum).
The increase in fluorescence intensity with PLTP (Rabbit Serum) is
usually 1.5-2.0 fold over blank (Organic solvent may increase
background readings and therefore proper control may be needed if
the testing inhibitor is prepared in organic solvent).
5. Data Analysis
Compare the fluorescence intensity of the testing inhibitor with
positive control to determine the inhibition efficiency of the testing
inhibitors.
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6. Troubleshooting
Problem
Reason
Solution
Assay not
working
Assay buffer at
wrong temperature
Assay buffer must not be chilled
- needs to be at RT
Protocol step missed
Plate read at
incorrect wavelength
Unsuitable microtiter
plate for assay
Unexpected
results
Re-read and follow the protocol
exactly
Ensure you are using
appropriate reader and filter
settings (refer to datasheet)
Fluorescence: Black plates
(clear bottoms)
Luminescence: White plates
Colorimetry: Clear plates
If critical, datasheet will indicate
whether to use flat- or U-shaped
wells
Measured at wrong
wavelength
Use appropriate reader and filter
settings described in datasheet
Samples contain
impeding substances
Unsuitable sample
type
Sample readings are
outside linear range
Troubleshoot and also consider
deproteinizing samples
Use recommended samples
types as listed on the datasheet
Concentrate/ dilute samples to
be in linear range
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Problem
Reason
Solution
Samples
with
inconsistent
readings
Unsuitable sample
type
Refer to datasheet for details
about incompatible samples
Use the assay buffer provided
(or refer to datasheet for
instructions)
Samples prepared in
the wrong buffer
Samples not
deproteinized (if
indicated on
datasheet)
Cell/tissue samples
not sufficiently
homogenized
Too many freezethaw cycles
Samples contain
impeding substances
Samples are too old
or incorrectly stored
Lower/
Higher
readings in
samples
and
standards
Not fully thawed kit
components
Out-of-date kit or
incorrectly stored
reagents
Reagents sitting for
extended periods on
ice
Incorrect incubation
time/temperature
Incorrect amounts
used
Use the 10kDa spin column
(ab93349)
Increase sonication time/
number of strokes with the
Dounce homogenizer
Aliquot samples to reduce the
number of freeze-thaw cycles
Troubleshoot and also consider
deproteinizing samples
Use freshly made samples and
store at recommended
temperature until use
Wait for components to thaw
completely and gently mix prior
use
Always check expiry date and
store kit components as
recommended on the datasheet
Try to prepare a fresh reaction
mix prior to each use
Refer to datasheet for
recommended incubation time
and/or temperature
Check pipette is calibrated
correctly (always use smallest
volume pipette that can pipette
entire volume)
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Standard
curve is not
linear
Not fully thawed kit
components
Pipetting errors when
setting up the
standard curve
Incorrect pipetting
when preparing the
reaction mix
Air bubbles in wells
Concentration of
standard stock
incorrect
Errors in standard
curve calculations
Use of other
reagents than those
provided with the kit
Wait for components to thaw
completely and gently mix prior
use
Try not to pipette too small
volumes
Always prepare a master mix
Air bubbles will interfere with
readings; try to avoid producing
air bubbles and always remove
bubbles prior to reading plates
Recheck datasheet for
recommended concentrations of
standard stocks
Refer to datasheet and re-check
the calculations
Use fresh components from the
same kit
For further technical questions please do not hesitate to
contact us by email (technical@abcam.com) or phone (select
“contact us” on www.abcam.com for the phone number for
your region).
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UK, EU and ROW
Email: technical@abcam.com
Tel: +44 (0)1223 696000
www.abcam.com
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Email: us.technical@abcam.com
Tel: 888-77-ABCAM (22226)
www.abcam.com
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Email: hk.technical@abcam.com
Tel: 108008523689 (中國聯通)
www.abcam.cn
Japan
Email: technical@abcam.co.jp
Tel: +81-(0)3-6231-0940
www.abcam.co.jp
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All information / detail is correct at time of going to print.