ab170965
Enterokinase Inhibitor
Screening Kit
(Fluorometric)
Instructions for Use
For screening Enterokinase inhibitors.
This product is for research use only and is not
intended for diagnostic use.
Version 1 Last Updated 19 March 2013
Table of Contents
1.
Overview
2
2.
Protocol Summary
3
3.
Kit Components
4
4.
Storage
4
5.
Additional Materials Required
5
6.
Assay Protocol
5
7.
Data Analysis
7
8.
Troubleshooting
8
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1. Overview
Enteropeptidase (Enterokinase, EC 3.4.21.9) is a serine protease
involved in activation of trypsinogen to trypsin, which in turn results
in the activation of various digestive enzymes. It recognizes a highly
specific amino acid sequence ‘DDDDK’ and cleaves after the lysine
residue. High specific activity of Enteropeptidase has been utilized in
cleaving a variety of native or fusion proteins containing the above
recognition.
Abcam's Enterokinase Inhibitor Screening Kit (Fluorometric) utilizes
a peptide substrate containing the Enteropeptidase recognition
sequence along with a fluorescent label ‘AFC’. Enteropeptidase
catalyzes the cleavage of this substrate and releases the AFC
molecule, which can be easily quantified by measuring its
fluorescence at Ex/Em = 380/500 nm. In the presence of potent
Enteropeptidase inhibitor, the hydrolyzation of the substrate will be
impeded. The kit provides a rapid, simple & reliable test for
screening potential inhibitors of Enteropeptidase.
2
2. Protocol Summary
Reagent Preparation
Sample Preparation
Positive Control
Reaction Mix
Measurement and Calculation
3
3. Kit Components
Item
Quantity
Storage
upon
arrival
Storage after
use/
reconstitution
0.1 mL
-20°C
-20°C
Enteropeptidase Assay Buffer
20 mL
-20°C
-20°C
Enteropeptidase Substrate
0.2 mL
-20°C
-20°C
Human Enteropeptidase
0.17 mL
-20°C
-80°C
Enteropeptidase-specific Inhibitor
[Aprotinin] (0.6 mM)
4. Storage
Store the kit at -20°C and protect from light. Please read the entire
protocol before performing the assay. Avoid repeated freeze/thaw
cycles.
Warm assay buffer to room temperature before use. Briefly
centrifuge all small vials at low speed prior to opening (high speed
not ideal for enzymes).
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5. Additional Materials Required

96-well clear plate with flat bottoms (white plates preferred
for this assay)

Multi-well spectrophotometer (ELISA reader)
6. Assay Protocol
A. Reagent Preparation
1. Human Enteropeptidase (Positive Control):
Reconstitute with 910 μL Enteropeptidase Assay Buffer to obtain
a solution of 1mU/µl. Aliquot & store at -80°C. Avoid repeated
freeze/thaw. Stable for 2 months at -80°C.
B. Enteropeptidase Inhibitor Screening Protocol
1. Enteropeptidase Enzyme Preparation:
For each well, prepare 50 μL of Enteropeptidase enzyme
solution.
40 μL Enteropeptidase Assay Buffer
10 μL Enteropeptidase enzyme (1mU/µl)
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2. Screening compounds, inhibitor control & blank control
preparations:
Dissolve candidate inhibitors into proper solvent. Dilute to 4X the
desired test concentration with Enteropeptidease Assay Buffer.
Add 25 μL diluted test inhibitors or Assay Buffer into
Enteropeptidase enzyme wells as sample screen [S] or Enzyme
Control [EC] (no inhibitor). For Inhibitor Control, add 10 ul
Enteropeptidease-specific inhibitor & 15 μL Enteropeptidase
Assay Buffer to Enteropeptidase enzyme well(s). Incubate at
room temperature for 10-15 min.
3. Enteropeptidase substrate preparation:
For each well, prepare 25 μL of the substrate solution.
23 μL of Enteropeptidase Assay Buffer
2 μL of Enteropeptidase Substrate
Mix & add 25 µl of Enteropeptidase Substrate solution into each
well. Mix well.
4. Measurement
Measure the fluorescence in a kinetic mode for 30-60 min
(Ex/Em = 380/500 nm). Choose two time points (T1 & T2) in the
linear range of the plot and obtain the corresponding values for
the fluorescence (RFU1 and RFU2).
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7. Data Analysis
Calculation: Calculate the slope for all samples, including Enzyme
Control (EC), by dividing the net ΔRFU (RFU2-RFU1) values with the
time ΔT (T2-T1).
‒ 𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒 × 100
% 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝐸𝐶
𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝐸𝐶
Figure 1: Inhibition of Enteropeptidase activity by Enteropeptidasespecific Inhibitor, Aprotinin. Assay was performed following kit
protocol.
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8. 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
Samples
with
inconsistent
readings
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
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
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Problem
Reason
Solution
Cell/ tissue samples
not sufficiently
homogenized
Too many freezethaw cycles
Samples contain
impeding substances
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
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
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
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)
Wait for components to thaw
completely and gently mix prior
use
Try not to pipette too small
volumes
Always prepare a master mix
9
Problem
Reason
Air bubbles in wells
Concentration of
standard stock
incorrect
Errors in standard
curve calculations
Use of other
reagents than those
provided with the kit
Solution
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
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All information / detail is correct at time of going to print.
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Copyright © 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.