Clarity™ Western
ECL Substrate
Instruction Manual
Catalog # Description
170-5060 Clarity Western ECL Substrate, 200 ml
170-5061 Clarity Western ECL Substrate, 500 ml
Imaging
Table of Contents
Introduction 1
Quick Start Protocol 1
Background on Chemiluminescence 2
Antibody Incubation 2
Detailed Assay Procedure 5
Example Western Protocol 5
Immunodetection 5
Chemiluminescent Development 6
Troubleshooting 8
Ordering Information 11
Introduction
The Bio-Rad Clarity™ western ECL substrate is compatible with
any HRP-conjugate secondary detection reagent and ideal
for both digital and lm-based imaging. The Clarity substrate
provides excellent sensitivity with an extremely long signal
duration that allows re-imaging without loss of signal. In addition,
Clarity substrate is formulated to exhibit very low background
levels that yield exceptionally clear images. The combination of
bright, long signal, and low background makes Clarity substrate
the perfect choice for most blotting applications.
Quick Start Protocol
1. After immunodetection, keep the membrane moist in wash
buffer as you prepare the substrate mixture.
2. Mix substrate kit components in a 1:1 ratio. Prepare 0.1 ml
of solution/cm2 of membrane.
• For a mini-sized membrane
(7 x 8.5 cm), 7 ml of solution is sufcient
• For a midi-sized membrane
(8.5 x 13.5 cm), 12 ml of solution is sufcient
3. Place the membrane protein side up on a clear surface.
• Add substrate to the blot and incubate for 5 min
4. Image the membrane with a digital imager or by exposing to
X-ray lm.
Storage Conditions
Kit components are stable for at least one year at room
temperature. Freezing will not adversely affect kit performance
but do not expose to multiple freeze/thaw cycles.
Technical Support: 1-800-424-6723 • www.bio-rad.com 1
Background on Chemiluminescence
Chemiluminescence is a chemical reaction that produces light
and has become a common detection method for western
blotting because of its high sensitivity. With chemiluminescent
western blots, a secondary antibody is conjugated to the enzyme
horseradish peroxidase (HRP). Once the secondary reagent is
bound to the target protein on the membrane, the membrane
is incubated with a solution containing the chemiluminescent
substrate (Fig. 1). In the presence of peroxide, the HRP enzymes
catalyzes the oxidation of luminol, which then generates light (Fig.
2). An enhancer is included in the substrate solution to increase
the longevity and intensity of the emitted light. Depending on the
substrate and enhancer formulation, the half-life of the
light-generating reaction can range from a few minutes to over
an hour. This light resulting from this reaction can be detected
with either lm or a digital imaging system. When combined with
a Bio-Rad digital imager such as the ChemiDoc™ MP, the
Bio-Rad Clarity substrate produces clear digital images that can
be directly used for analysis or publication.
Antibody Incubations
A typical immunodetection experiment system utilizes two sets of
antibodies.
• The primary antibody, which is directed against the
target protein (Antigen)
• The secondary reagent, in this case an antibody that
recognizes and binds to the primary antibody; it is
conjugated to an enzyme such as HRP, which will
convert the substrate into light, which is then detected
by an imager of film
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Fig. 1. Specific enzymatic detection of membrane-bound
antigens.
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Fig. 2
Chemiluminescence
detection by HRP.
The secondary antibody
is linked to an enzyme,
which catalyzes a reaction
leading to light emission.
Luminol oxidized by
HRP in the presence
of a peroxide leads to
the formulation of a
3-aminophthalate dianion
and the release of light.
Detailed Assay Procedure
Example Western Protocol
Materials
• PVDF, LF PVDF, or nitrocellulose membrane with transferred
proteins
• Blocking buffer (Tris-buffered saline (TBS) or phosphatebuffered saline (PBS) with 0.05% Tween-20 and 1–6% of
a blocking reagent, typically BSA, gelatin, casein, or nonfat
dry milk
• Wash buffer (TBS or PBS with 0.05% Tween-20)
• Primary antibody, diluted in blocking buffer
• HRP-conjugated secondary reagent, such as goat
anti-rabbit or goat anti-mouse conjugated HRP, diluted in
wash buffer
Immunodetection
1. Wash buffer volumes should be at least 20 ml for mini blots
and 100 ml for midi blots. Block and antibody solution
volumes should be at least 10 ml for mini blots and 25 ml for
midi blots.
2. Equilibrate membrane with transferred proteins in wash
buffer for 3 minutes. Dried PVDF and LF PVDF membranes
should be briey re-wet in methanol prior to equilibration in
wash buffer.
3. Incubate the membrane, protein side up, in blocking buffer
for 1 hr with continuous agitation.
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4. Incubate the membrane in diluted primary antibody solution
for 1 hr with continuous agitation.
• Incubation in primary antibody may be carried out
overnight at 4°C
5. Wash the blot in wash buffer 5 times for 5 min each with
continuous agitation.
6. Incubate the blot in diluted secondary antibody solution for
1 hr with continuous agitation.
7. Wash the blot in wash buffer 6 times for 5 min each with
continuous agitation.
Chemiluminescent Development
1. Keep the membrane moist in wash buffer as you prepare
the substrate mixture.
• Do not allow the membrane to dry out during the
subsequent steps
2. Mix substrate kit components in a 1:1 ratio. Prepare 0.1 ml
of solution/cm2 of membrane.
• For a mini-sized membrane (7 x 8.5 cm), 7 ml of
solution is sufficient
• For a midi-sized membrane (8.5 x 13.5 cm), 12 ml of
solution is sufficient
3. Place the membrane, protein side up, on a clear surface.
• Add substrate to the blot and incubate for 5 min
• Ensure the surface of the blot is completely covered
with substrate with no air bubbles
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4. Remove the membrane from the substrate solution and
drain off excess.
5. Place membrane in a plastic sheet protector or in plastic
wrap to prevent the membrane from drying.
6. Image the membrane with a digital imager or by exposing to
X-ray lm.
• If switching to Bio-Rad Clarity from another chemi
substrate, optimal exposure times may be different.
Refer to the Substrate Transition Guide below when
switching substrates
Transition Guide. To transition to Clarity, use the following
guidelines to adjust exposure times.
Current Substrate Exposure Using Clarity
GE ECL/Pierce ECL Decrease 20-fold*
SuperSignal West Pico Decrease 5-fold*
Bio-Rad® Immun-Star™ HRP Decrease 5-fold*
Pierce ECL Plus Equal
Bio-Rad® Immun-Star™ WesternC™Equal
SuperSignal West Dura Equal
ECL Prime Increase 2-fold
SuperSignal West Femto Increase 2-fold
ECL Select Increase 2-fold
*Or decrease antibody dilution by 2- to 5-fold.
ECL Select is a trademark of GE Healthcare UK limited.
SuperSignal is a trademark of Thermo Fisher Scientic Inc.
Tween is a trademark of ICI Americas, Inc.
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Troubleshooting
Problem Cause Solution
High
background
Areas of no
signal within
a band
(a donut
appearance)
Blocking was
incomplete
Washing was
insufficient
The primary
or secondary
antibody
was too
concentrated
Localized
substrate
depletion
• Increase the
concentration of blocking
agent or increase
blocking duration
• Match the blocker to
the membrane. For
example, gelatin may give
poor results on PVDF
membranes
• Increase the number,
duration, or stringency of
the washes
• Decrease antibody
concentrations
• Perform a dot-blot
experiment to
optimize the working
concentrations
• Bands on the blot with
high protein amounts
will lead to excessive
local concentrations of
peroxidase conjugate.
This may lead to localized
substrate depletion. Use
lower concentrations of
primary and secondary
antibody
• Load less sample
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Troubleshooting, continued
Problem Cause Solution
No reaction
or weak
signal
Proteins may
be washed
from the
membrane
during assays
• Reduce the number or
stringency of washes
Antigen
binding to the
membrane
was
insufficient
• Decrease antibody
concentrations
• Stain the gel after
transfer or use
prestained standards to
assess transfer efficiency
• Some total protein
stains (such as amido
black and colloidal gold)
interfere with antibody
recognition of the
antigen. Do not use a
total protein stain, or use
a different stain
• Optimize the blocking
reagent. Some blocking
reagents (such as
nonfat dry milk) provide
high stringency at the
expense of sensitivity.
Others (such as BSA)
offer comparatively high
sensitivity at the expense
of higher background or
nonspecific binding
Technical Support: 1-800-424-6723 • www.bio-rad.com 9
Troubleshooting, continued
Problem Cause Solution
Poor antibody
binding to the
antigen
Insuffecient
reagent
volume
• Detergents may affect
the binding of some
antibodies. Eliminate or
reduce their amount from
the assay
• Increase the antibody
incubation times
• Use additional volumes of
blocking, antibody, and
wash solutions
The enzyme
conjugate was
inactive
• Test the reagent for
activity
• Horseradish peroxidase
is most active at optimal
pH. Ensure excess wash
buffer is removed from
the membrane before
application of substrate
• Sodium azide is a potent
inhibitor of horseradish
peroxidase. Eliminate
from antibody stocks
Blank spots
in areas of
membrane
that should
have signal
The
membrane
was allowed
to dry during
handling
• Ensure that no air
bubbles were present
during assembly of
transfer stack
• Ensure that warm
membranes are not
allowed to dry after
transfer
10 Technical Support: 1-800-424-6723 • www.bio-rad.com
Ordering Information
Catalog Product
170-5060 Clarity™ Western ECL Substrate, 200 ml
size contains Clarity western peroxide reagent,
100 ml, and Clarity western luminol/enhancer
reagent,100 ml
170-5061 Clarity Western ECL Substrate, 500 ml size
contains Clarity western peroxide reagent,
250 ml, and Clarity western luminol/enhancer
reagent, 250 ml
Technical Support: 1-800-424-6723 • www.bio-rad.com 11
Bio-Rad
Laboratories, Inc.
Life Science
Group
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