Bio-Rad Clarity Western ECL Substrate User Manual

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 sufcient

• For a midi-sized membrane

(8.5 x 13.5 cm), 12 ml of solution is sufcient

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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4 Technical Support: 1-800-424-6723 • www.bio-rad.com

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 briey 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

6 Technical Support: 1-800-424-6723 • www.bio-rad.com

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 Scientic 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

8 Technical Support: 1-800-424-6723 • www.bio-rad.com

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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