Version 2a Last updated 15 February 2021

ab239722
Pepsin/Pepsinogen
Assay Kit
(Fluorometric)

For the measurement of pepsin in biological samples.

This product is for research use only and is not intended for
diagnostic use.

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

Table of Contents

1. Overview 3
2. Materials Supplied and Storage 4
3. Materials Required, Not Supplied 5
4. General guidelines, precautions, and troubleshooting 6
5. Reagent Preparation 7
6. Standard Preparation 8
7. Sample Preparation 9
8. Assay Procedure 10
9. Data Analysis 12
10. Typical Data 13
11. Notes 15

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

1. Overview

Pepsin/Pepsinogen Assay Kit (Fluorometric) (ab238722) is a

homogenous assay that allows for quantification of pepsin activity in
gastric tissues (stomach, duodenum etc.) and various biological
fluids (serum/plasma, gastric juice, vomit). The assay utilizes a
synthetic peptide substrate bearing both a fluorophore and a
fluorescence quencher. Upon cleavage by pepsin, the fluorophore-
bearing peptide fragment is unquenched to produce a bright
fluorescent signal (Ex/Em = 328/418 nm). Lysosomal aspartic
proteases in the peptidase A1 family (Cathepsin D and E) do not
interfere with the assay. The assay is rapid, simple to perform and is
vastly more sensitive than the classical hemoglobin degradation
assay, with a detection limit of 500 μU pepsin activity per well.

Pepsin / acidic conditions

Peptide substrate Fluorescent peptide fragment
(Quenched) (Ex/Em = 328/418 nm)

Prepare tissue / serum / plasma sample and Pepsin Positive Control.

Prepare Standard Curve.

Prepare Reaction Mix, Inhibitor Reaction Mix, Background Control

Mix and Positive Control Reaction Mix.

Start reaction by adding 20 μL diluted Substrate working solution to

each well (apart from Standard Curve).

Measure the fluorescence (Ex/Em = 328/418 nm) of all sample wells

in kinetic mode for 60 minutes at 37°C.

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

 2. Materials Supplied and Storage

Store kit at -20°C in the dark immediately on receipt and check

below for storage for individual components. Kit can be stored for 1
year from receipt, if components have not been reconstituted.

Avoid repeated freeze-thaws of reagents.

Storage Storage
temperatur temperatur
Item Quantity
e (before e (after
prep) prep)
Pepsin Assay Buffer 25 mL -20°C -20°C
Pepsin Substrate 200 μL -20°C -20°C

Pepsin Inhibitor (Pepstatin A) 20 μL -20°C -20°C

Pepsin Positive Control 1 vial -20°C -20°C

MCA Standard 25 μL -20°C -20°C

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

3. Materials Required, Not Supplied

These materials are not included in the kit, but will be required to
successfully perform this assay:
 Microplate reader capable of measuring fluorescence at
Ex/Em = 328/418 nm.
 White 96-well plate with clear flat bottom.

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

4. General guidelines, precautions, and troubleshooting

Please observe safe laboratory practice and consult the safety

datasheet.
For general guidelines, precautions, limitations on the use of our
assay kits and general assay troubleshooting tips, particularly for first
time users, please consult our guide:
www.abcam.com/assaykitguidelines
For typical data produced using the assay, please see the assay kit
datasheet on our website.

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

5. Reagent Preparation

Briefly centrifuge small vials at low speed prior to opening.

5.1 Pepsin Substrate

Provided as a stock solution in DMSO. Divide into aliquots and
store at -20°C, protected from light. Prior to use, warm solution
to room temperature and vortex thoroughly.
5.2 Pepsin Inhibitor (Pepstatin A)
Provided as a 1 mM stock solution in DMSO. Prior to use, warm
solution to room temperature and vortex thoroughly. Store at -
20°C, stable for at least 3 freeze/thaw cycles.
5.3 Pepsin Positive Control
Reconstitute with 110 μL ddH2O. Divide into aliquots and store
at -20°C. Avoid repeated freeze/thaw cycles
5.4 MCA Standard
Provided as a 1 mM stock solution in DMSO. Warm solution to
room temperature prior to use. Store at -20°C, protected from
light. Stable for at least 3 freeze/thaw cycles.
5.5 Pepsin Assay Buffer
Ready to use as supplied. Warm to room temperature prior to
use.

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

6. Standard Preparation

 Always prepare a fresh set of standards for every use.

 Discard working standard dilutions after use as they do not store
well.

6.1 Prepare a 50 µM working solution of MCA by adding 5 µL of

the 1 mM MCA Standard to 95 µl of Pepsin Assay Buffer.
6.2 Add 0, 2, 4, 6, 8, and 10 µL of the 50 µM working solution into a
series of wells in a white 96-well plate, generating 0, 100, 200,
300, 400 and 500 pmol of MCA/well. Adjust the volume of all
standard curve wells (including the 0 pmol/well reagent blank)
to 100 µL with Pepsin Assay Buffer.

MCA Pepsin Final

MCA
Standard Assay volume
Standard Standard
# Buffer standard in
(µL) (pmol/well)
(µL) well (µL)
1 20 180 200 500
2 16 184 200 400
3 12 188 200 300
4 8 192 200 200
5 4 196 200 100
6 0 200 200 0

Each dilution has enough standard to set up duplicate readings (2 x

100 µL).

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

7. Sample Preparation

General sample information:

We recommend performing several dilutions of your sample to
ensure the readings are within the standard value range.
We recommend that you use fresh samples for the most
reproducible assay.

7.1 Mammalian tissue samples:

1. Homogenize fresh or frozen tissue with Pepsin Assay Buffer (100 µL
per ~10 mg of wet tissue) on ice and vortex thoroughly.
Centrifuge the homogenate at 4°C for 10 minutes at 10,000 x g.
2. Transfer the clarified supernatant to a fresh pre-chilled microfuge
tube and keep on ice during use. Add 5-20 μL of clarified
homogenate to desired well(s) in a white, flat-bottomed 96-well
plate.

7.2 Serum and plasma samples:

1. Collect serum or plasma samples by standard methods. Samples
exhibiting lipemia or excessive turbidity should be clarified by
centrifugation at 10,000 x g for 5 minutes in order to separate
lipid globules.
2. Add 5-20 µL of undiluted serum/plasma sample to desired
well(s).

7.3 Pepsin positive control:

1. Dilute the reconstituted Pepsin Positive Control at 1:10 ratio by
mixing 10 µL of the reconstituted stock with 90 µL of Pepsin Assay
Buffer.
2. Incubate the diluted Pepsin Positive Control solution at room
temperature for 5 minutes to allow for acid-mediated pepsin
auto-activation, then add 10 µL of diluted Pepsin Positive Control
solution to desired well(s).

Note: Pepsin undergoes autolysis (self-cleavage) in solution at pH

values ≤ 4. To prevent pepsin autolysis, we recommend storing tissue
homogenate samples at -80°C if they are to be used in future
experiments.

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

8. Assay Procedure

 Assay all standards, controls and samples in duplicate.

8.1 Prepare assay reaction wells according to the table below. In

addition to the test sample wells, prepare a background
control (substrate only) well to correct for potential non-
enzymatic substrate hydrolysis.
8.2 For further verification of pepsin activity, you may prepare
inhibitor control wells (sample + 1 µM Pepstatin A). To prepare
inhibitor control wells, dilute the Pepsin Inhibitor (Pepstatin A)
stock solution at 1:100 ratio in Pepsin Assay Buffer to produce a
10 µM working solution and add 10 μL of the working solution
per well.

Plus Background Positive

Reaction
Component Inhibitor Control Mix Control
Mix (µL)
Mix (µL) (µL) (µL)
Test sample 5-20 5-20 - -
Diluted Pepsin
- - - 10
Positive control
Pepstatin A (10
- 10 - -
µM)
Pepsin Assay
to 80 µL to 80 µL 80 70
Buffer

8.3 Preincubate the plate for 10 min at 37°C to allow sample

temperature to equilibrate. During the preincubation, prepare
Pepsin Substrate working solution by diluting the Pepsin
Substrate stock solution with Pepsin Assay Buffer at a 1:10 ratio.
Prepare 20 µL of substrate working solution for each reaction
to be performed (for example, for 10 reaction wells, mix 20 µL
of Pepsin Substrate stock with 180 µL Pepsin Assay Buffer).
8.4 Start the reaction by adding 20 µL of the diluted substrate
working solution to each reaction well, yielding a final volume
of 100 µL per well.

Note: Do not add Pepsin Substrate solution to the MCA Standard

Curve wells.

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

8.5 Measure the fluorescence (Ex/Em = 328/418 nm) of all sample
wells in kinetic mode for 60 min at 37°C. We strongly
recommend reading in kinetic mode in order to ensure that
the measurements recorded are within the linear range of the
reaction. Ideal measurement time for the linear range may
vary depending upon the sample.

Note: The MCA Standard curve wells may be read in endpoint

mode (Ex/Em = 328/418 nm).

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

9. Data Analysis

Samples producing signals greater than that of the highest standard

should be further diluted in appropriate buffer and reanalyzed, then
multiply the concentration found by the appropriate dilution factor.

1. Average the duplicate reading for each standard, control and

sample.
2. For the MCA Standard curve, subtract the fluorescence
obtained for the reagent blank (0 pmol/well standard) from all
of the standard readings, plot the background-subtracted
values and calculate the slope of the standard curve.
3. For all reaction wells (including background control), choose
two time points (t1 and t2) in the linear phase of the reaction
progress curves, obtain the corresponding fluorescence values
at those points (RFU1 and RFU2) and determine the change in
fluorescence over the time interval: ∆F = RFU2 – RFU1.
4. If the ∆F value for the background control well is significant, it
should be subtracted from each test sample to obtain the
corrected fluorescence: FC = ∆Fsample – ∆FBC. If ∆FBC is negative,
background subtraction should be ignored: FC = ∆Fsample.
5. Apply the FC values to the MCA Standard curve to get B pmol of
unquenched MCA-peptide in the well.

B
Sample Pepsin Activity = *D
∆T * P = pmol/min/(mL or mg)

Where:
B = amount of peptide substrate cleaved, calculated from the
Standard Curve (in pmol).
T = linear phase reaction time t1 – t2 (in minutes).
P = the amount of sample added to the well (in ml of biological fluid
or mg of protein)
D = sample dilution factor applied (if applicable, D = 1 for undiluted
samples).

Pepsin Unit Definition: One unit of pepsin activity is the amount of

enzyme that generates 1 μmole of unquenched 7-

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

methoxycoumarin-4-acetate (MCA) per min by hydrolysis of 1 μmole
peptide substrate at 37°C and pH 2.

10. Typical Data

Data provided for demonstration purposes only.

Figure 1. MCA standard curve.

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

Figure 2. Kinetics of Pepsin Substrate metabolism by pig gastric mucosal
pepsin (3.33 ng purified enzyme) and specificity of substrate metabolism by
pepsin versus other aspartic proteases. The acid-activated proteases
Cathepsin D and E exhibit minimal assay interference, even when present at
≥300-fold excess by mass.

Figure 3. Estimation of pepsinogen activity in pooled normal human serum

and single-donor serum from a gastric ulcer patient with confirmed H. Pylori
infection (each 10 μL of undiluted serum). Data are mean ± SEM of 3
replicates.

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

11. Notes

ab239722 Pepsin/Pepsinogen Assay Kit (Fluorometric)

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