I
N
N
O
V
A
T
I
O
N
S
F
O
R
U
A review of radioactive and non-radioactive-based
techniques used in life science applications:
Part I Blotting techniques
J. Osborn
Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK
L
A
B
E
L
L
I
N
G
A
N
D
D
E
T
E
C
T
I
O
N
screening, DNA sequencing and fragment analysis, and
in situ hybridization. In Part I, labelling techniques used
in blotting applications are discussed. The complete
series is available on-line at www.apbiotech.com/lsn6-7.
In recent years, fluorescence, chemifluorescence and
chemiluminescence have emerged as alternative
technologies to the traditional radioisotope-based
systems still widely used in many life science research
applications. Convenience, speed and safety are
important factors in deciding which label to use,
however there are various areas whereby
radioisotopes continue to offer significant
advantages over non-radioactive methods.
M
I
N
N
O
Introduction
Techniques based on the use of radioactive labels offer
robust and reproducible protocols in applications that
require ultimate sensitivity and/or resolution. Quantification of results is possible following exposure of
signal to autoradiography film or reusable storage
phosphor screens in automated imaging systems.
However, automated liquid handling of radioactivity
is difficult due to the need for safe handling and
disposal of radioactivity, and is generally limited to
manual, low-throughput applications.
For most Northern and Southern blotting applications,
sensitivity is considered to be of great importance.
Uniform labelling methods, such as random-prime, nick
translation, and transcription from cloned promoters
offer rapid probe labelling and high label density, and
are therefore most frequently used. A variety of DNA
labelling systems offer simple and rapid probe labelling
times from as little as 5 min compared with 30 min for
most non-radioactive systems (Table 1).
Chemiluminescence offers a highly sensitive alternative
to radioactivity, but low intensity signal means it is not
amenable to automation and quantification is difficult
due to the narrow linear range of the signal produced.
Major advances in fluorescent and chemifluorescent
dye chemistries include the development of fully
automated instrumentation and sophisticated
imaging systems capable of powerful data manipulation and analysis. These multi-modal platforms have
presented researchers with a range of sensitive and
versatile investigative tools for an increasingly diverse
range of application areas while providing a highthroughput solution for the increasing demands
of the modern laboratory.
32P
Nucleotides labelled with
enable high specific
activity probes up to 2 × 109 dpm/µg to be produced
that are capable of detecting down to 10 fg of target
DNA (Table 1). They are more sensitive than any
chemiluminescence or chemifluorescence-based
labelling system making 32P the best label for detection
of rare sequences.
In this four-part series, the use of radioactive and
non-radioactive labelling and detection methods is
examined for blotting techniques, high-throughput
Advances in autoradiography film emulsion have
further improved the maximum sensitivity that can be
obtained with high energy-emitting radiolabels such as
A
T
I
O
N
S
F
O
R
U
Table 1. Comparison of radioactive and non-radioactive nucleic acid labelling and detection systems.
Labelling and detection system
Sensitivity
Application
Probe labelling
time
Time from hybridization
to detection
32P
Down to 10 fg
All high sensitivity applications
5 min to 3 h
On film 1 h to 1 week
AlkPhos Direct
60 fg
All high sensitivity applications
30 min
1h
ECL Direct
0.5 pg
High target applications
20 min
1–2 h
Gene Images Random-Prime with
CDP-Star Detection
50 fg
High sensitivity Northern blots
30 min
3h
Gene Images 3'-Oligolabelling with CDP-Star
0.1 pg
Oligonucleotide screening with
stringency control
30 min
3h
Gene Images Random-Prime with ECF
0.25 pg
Quantification
30 min
3h
Gene Images 3'-Oligolabelling with ECF
120 pg
Quantification
30 min
3h
ECL Random-Prime
0.5 pg
Medium target Southern blots
with DNA probes
30 min
3h
ECL 3'-End Labelling
0.2 pg
Medium to high target Southern
blots with oligonucleotide probes
30 min
3h
Use of radioactivity in nucleic acid applications
Both radioactive and non-radioactive probes are used
extensively for a wide range of Northern and Southern
blotting applications. The choice of label type is
generally dependent on a number of considerations
including sensitivity and resolution required. Uniformly labelled, radioactive probes can be rapidly
prepared using a variety of labelling systems to produce high specific activity DNA probes. However, the
short shelf-life of radioactive labels plus the licensing
and safety issues associated with radioactivity, mean
the convenience, speed and sensitivity available with
non-radioactive methods form a significant part of the
decision making process.
V
H
14
C
35
S
33
P
fluorescein antibody conjugated to alkaline phosphatase (AP) enzyme (Fig 2). Fluorescein-labelled
DNA is stable under standard hybridization conditions and, as with radiolabelled probes, the stringency
of hybridization can be controlled either with temperature or salt concentration. Chemiluminescent detection using CDP-Star™ as the substrate for the enzyme
enables as little as 50 fg of target to be routinely
detected in a human genomic Southern blot, making
it a sensitive alternative to 32P (Table 1).
BioMax MS Film and MS Screen
BioMax MR Film
3
32
P
32
P&
Overcoat
(Includes
Protective
Dye)
125
I
Sample
7 mil
Estar Base
Emulsion
Layer
Intensifying
Screen
Fig 1. Principle of direct autoradiography and normal intensifying
screens.
32P.
The extremely light-sensitive emulsion of Kodak’s
BioMax MS film reduces 4-day exposures to 1 day and
overnight exposures to 4 hours when used with Kodak
BioMax MS or BioMax TranScreen™ HE intensifying
screens (Fig 1). This represents a four-fold increase in
the speed of detection of 32P-labelled probes when
compared to normal film/screen combinations.
Blots hybridized using Gene Images Random-Prime
Labelling and 3'-Oligolabelling modules can also be
detected with ECF substrate. This non-fluorescent
substrate is catalysed by AP to produce a chemifluorescent signal that accumulates over time. Low
sensitivity applications yield results after 1 h while
high sensitivity applications usually require overnight
incubation. Quantification of low sensitivity applications such as dot and slot blots is possible by using
ECF detection and results can be analysed on a
fluorescence scanner such as Typhoon, Storm,
FluorImager, or ImageMaster VDS-CL systems.
Use of non-radioactive methods in
nucleic acid applications
For many years the use of radiolabels was the method
of choice for nucleic acid labelling and detection.
However, significant improvements to the sensitivity
that can be achieved with non-radioactive labelling
and detection systems have lead to the evolution of a
comprehensive range of products that offer rapid,
non-destructive alternatives to the use of traditional
radioactive techniques.
Direct labelling methods are alternatives to indirect
methods and offer significant improvements in speed
and convenience without compromising sensitivity.
There are an increasing number of non-radioactive
labels that are available for direct labelling. CyDyes
are part of a range of fluorescent dyes that can be
either coupled directly to nucleotides or conjugated
to antibodies. Fluorescently labelled nucleotides are
also available and these can be incorporated into
DNA probes by nick translation or random-priming.
Non-radioactive labelling methods are separated into
two different approaches, indirect and direct.
Indirect, hapten-based labelling kits, such as Gene
Images™ Labelling and Detection Systems, introduce
nucleotides tagged with fluorescein into the probe.
These are then detected with a highly specific anti-
Life Science News 6, 2000 Amersham Pharmacia Biotech
Life Science News 6, 2000 Amersham Pharmacia Biotech
22
23
M
I
N
N
O
V
A
T
I
O
N
S
F
O
R
U
A review of radioactive and non-radioactive-based
techniques used in life science applications:
Part I Blotting techniques
J. Osborn
Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK
L
A
B
E
L
L
I
N
G
A
N
D
D
E
T
E
C
T
I
O
N
screening, DNA sequencing and fragment analysis, and
in situ hybridization. In Part I, labelling techniques used
in blotting applications are discussed. The complete
series is available on-line at www.apbiotech.com/lsn6-7.
In recent years, fluorescence, chemifluorescence and
chemiluminescence have emerged as alternative
technologies to the traditional radioisotope-based
systems still widely used in many life science research
applications. Convenience, speed and safety are
important factors in deciding which label to use,
however there are various areas whereby
radioisotopes continue to offer significant
advantages over non-radioactive methods.
M
I
N
N
O
Introduction
Techniques based on the use of radioactive labels offer
robust and reproducible protocols in applications that
require ultimate sensitivity and/or resolution. Quantification of results is possible following exposure of
signal to autoradiography film or reusable storage
phosphor screens in automated imaging systems.
However, automated liquid handling of radioactivity
is difficult due to the need for safe handling and
disposal of radioactivity, and is generally limited to
manual, low-throughput applications.
For most Northern and Southern blotting applications,
sensitivity is considered to be of great importance.
Uniform labelling methods, such as random-prime, nick
translation, and transcription from cloned promoters
offer rapid probe labelling and high label density, and
are therefore most frequently used. A variety of DNA
labelling systems offer simple and rapid probe labelling
times from as little as 5 min compared with 30 min for
most non-radioactive systems (Table 1).
Chemiluminescence offers a highly sensitive alternative
to radioactivity, but low intensity signal means it is not
amenable to automation and quantification is difficult
due to the narrow linear range of the signal produced.
Major advances in fluorescent and chemifluorescent
dye chemistries include the development of fully
automated instrumentation and sophisticated
imaging systems capable of powerful data manipulation and analysis. These multi-modal platforms have
presented researchers with a range of sensitive and
versatile investigative tools for an increasingly diverse
range of application areas while providing a highthroughput solution for the increasing demands
of the modern laboratory.
32P
Nucleotides labelled with
enable high specific
activity probes up to 2 × 109 dpm/µg to be produced
that are capable of detecting down to 10 fg of target
DNA (Table 1). They are more sensitive than any
chemiluminescence or chemifluorescence-based
labelling system making 32P the best label for detection
of rare sequences.
In this four-part series, the use of radioactive and
non-radioactive labelling and detection methods is
examined for blotting techniques, high-throughput
Advances in autoradiography film emulsion have
further improved the maximum sensitivity that can be
obtained with high energy-emitting radiolabels such as
A
T
I
O
N
S
F
O
R
U
Table 1. Comparison of radioactive and non-radioactive nucleic acid labelling and detection systems.
Labelling and detection system
Sensitivity
Application
Probe labelling
time
Time from hybridization
to detection
32P
Down to 10 fg
All high sensitivity applications
5 min to 3 h
On film 1 h to 1 week
AlkPhos Direct
60 fg
All high sensitivity applications
30 min
1h
ECL Direct
0.5 pg
High target applications
20 min
1–2 h
Gene Images Random-Prime with
CDP-Star Detection
50 fg
High sensitivity Northern blots
30 min
3h
Gene Images 3'-Oligolabelling with CDP-Star
0.1 pg
Oligonucleotide screening with
stringency control
30 min
3h
Gene Images Random-Prime with ECF
0.25 pg
Quantification
30 min
3h
Gene Images 3'-Oligolabelling with ECF
12 pg
Quantification
30 min
3h
ECL Random-Prime
0.5 pg
Medium target Southern blots
with DNA probes
30 min
3h
ECL 3'-End Labelling
0.2 pg
Medium to high target Southern
blots with oligonucleotide probes
30 min
3h
Use of radioactivity in nucleic acid applications
Both radioactive and non-radioactive probes are used
extensively for a wide range of Northern and Southern
blotting applications. The choice of label type is
generally dependent on a number of considerations
including sensitivity and resolution required. Uniformly labelled, radioactive probes can be rapidly
prepared using a variety of labelling systems to produce high specific activity DNA probes. However, the
short shelf-life of radioactive labels plus the licensing
and safety issues associated with radioactivity, mean
the convenience, speed and sensitivity available with
non-radioactive methods form a significant part of the
decision making process.
V
H
14
C
35
S
33
P
fluorescein antibody conjugated to alkaline phosphatase (AP) enzyme (Fig 2). Fluorescein-labelled
DNA is stable under standard hybridization conditions and, as with radiolabelled probes, the stringency
of hybridization can be controlled either with temperature or salt concentration. Chemiluminescent detection using CDP-Star™ as the substrate for the enzyme
enables as little as 50 fg of target to be routinely
detected in a human genomic Southern blot, making
it a sensitive alternative to 32P (Table 1).
BioMax MS Film and MS Screen
BioMax MR Film
3
32
P
32
P&
Overcoat
(Includes
Protective
Dye)
125
I
Sample
7 mil
Estar Base
Emulsion
Layer
Intensifying
Screen
Fig 1. Principle of direct autoradiography and normal intensifying
screens.
32P.
The extremely light-sensitive emulsion of Kodak’s
BioMax MS film reduces 4-day exposures to 1 day and
overnight exposures to 4 hours when used with Kodak
BioMax MS or BioMax TranScreen™ HE intensifying
screens (Fig 1). This represents a four-fold increase in
the speed of detection of 32P-labelled probes when
compared to normal film/screen combinations.
Blots hybridized using Gene Images Random-Prime
Labelling and 3'-Oligolabelling modules can also be
detected with ECF substrate. This non-fluorescent
substrate is catalysed by AP to produce a chemifluorescent signal that accumulates over time. Low
sensitivity applications yield results after 1 h while
high sensitivity applications usually require overnight
incubation. Quantification of low sensitivity applications such as dot and slot blots is possible by using
ECF detection and results can be analysed on a
fluorescence scanner such as Typhoon, Storm,
FluorImager, or ImageMaster VDS-CL systems.
Use of non-radioactive methods in
nucleic acid applications
For many years the use of radiolabels was the method
of choice for nucleic acid labelling and detection.
However, significant improvements to the sensitivity
that can be achieved with non-radioactive labelling
and detection systems have lead to the evolution of a
comprehensive range of products that offer rapid,
non-destructive alternatives to the use of traditional
radioactive techniques.
Direct labelling methods are alternatives to indirect
methods and offer significant improvements in speed
and convenience without compromising sensitivity.
There are an increasing number of non-radioactive
labels that are available for direct labelling. CyDyes
are part of a range of fluorescent dyes that can be
either coupled directly to nucleotides or conjugated
to antibodies. Fluorescently labelled nucleotides are
also available and these can be incorporated into
DNA probes by nick translation or random-priming.
Non-radioactive labelling methods are separated into
two different approaches, indirect and direct.
Indirect, hapten-based labelling kits, such as Gene
Images™ Labelling and Detection Systems, introduce
nucleotides tagged with fluorescein into the probe.
These are then detected with a highly specific anti-
Life Science News 6, 2000 Amersham Pharmacia Biotech
Life Science News 6, 2000 Amersham Pharmacia Biotech
22
23
M
I
N
N
O
V
A
Substrate
T
I
O
S
F
O
R
U
M
I
N
N
O
V
A
T
I
O
platforms incorporating highly sensitive optic systems
such Typhoon 8600 and ImageMaster VDS-CL enable
the direct detection of chemiluminescence without the
need for intermediate exposures to films or screens.
However, unlike the signal produced by radioactive
probes, chemiluminescence is only linear over a narrow range and therefore offers limited quantification.
Light
Enzyme
Antibody
N
A
S
B
F
O
R
U
Fig 5. Target: mouse IgG slot blotted on to
PVDF (Hybond-P, RPN2020F):loadings:
doubling dilutions starting at 5 ng, detection: 1:2500 dilution of anti-mouse Ig HRP
conjugate (NA931) using (A) ECL Detection
Reagents (RPN2106) and (B) ECL Plus
Detection Reagents (RPN2132); exposure:on Hyperfilm ECL (RPN2103)
for 5 minutes.
Use of radioactivity in protein applications
Fluorescein
Probe
For detection of enzyme-labelled antibodies, the most
commonly used radiolabels are 125I and 35S, although
the range of radiolabelled antibodies is distinctly biased
towards 125I. This is largely due to the extensive use of
125I-labelled antibodies in clinical radioimmunoassays,
making them very accessible to researchers performing
Western blots. Furthermore, the signal produced by
125I and 35S is unaffected by enzyme, metal salts,
pH, or temperature, making them very useful for
new and uncharacterized systems often used during
life science research.
Target DNA
Membrane
Fig 2. Outline of indirect labelling method.
The fluorophores of hybridized probes can then be
visualized directly using systems such as Typhoon,
Storm, FluorImager, and ImageMaster VDS-CL.
Because different fluorophores are detected as different colours, it is possible to visualize two or more
different probes at one time, making fluorescence
multiplexing possible.
The signal produced by radiolabelled antibodies is
typically captured on autoradiographic film, providing
a permanent, hard-copy result. Multiple copies can be
produced from the same blot and results are easily
quantifiable by densitometry. However, filmless
autoradiography of radioactive Western blots is
possible through the use of re-usable, general-purpose
storage phosphor screens incorporated into image
analysis systems such as PhosphorImager™, Storm,
and Typhoon (Fig 4). These screens retain energy from
beta particles, X-rays and gamma rays and typically
50–90% less exposure time is required compared with
an equivalent exposure to conventional film.
With direct chemifluorescence labelling systems such
as AlkPhos Direct, AP is directly crosslinked to the
nucleic acid probe in a simple, 30 min reaction (Fig 3).
The probe is hybridized to the blot and incubated.
Detection is possible 1 h after hybridization. Direct
labelling saves 3–4 h compared to indirect labelling
methods (Fig 2). This is because the antibody conjugate incubation and associated blocking and washing
steps have been eliminated.
Results from fluorescent and chemifluorescent methods can be quantified using various imaging systems,
which allow filmless detection and subsequent image
analysis using powerful software packages including
ImageMaster™ and ImageQuant™.
Use of non-radioactive methods in
protein applications
Chemiluminescent detection systems were originally
designed for use with autoradiography film because
of the low levels of light produced. However, imaging
CDP-Star
or ECF substrate
N
Chemiluminescent and chemifluorescent methods for
the detection of enzyme-labelled antibodies have
largely replaced radioactivity in the immunodetection
of proteins. This is because non-radioactive systems are
highly sensitive, yield hard-copy results for subsequent
analysis after as little as 1min exposure to autoradiography film, and are safer to handle than radioisotopes.
Signal
ECL Western Blotting Analysis System is capable of
detecting < 1 pg of antigen on Hybond™ ECL membrane
making it suitable for routine Western blotting applications. Furthermore, the linear response exhibited by
Hyperfilm™ ECL to the signal means accurate quantification of proteins on ECL Western blots is possible.
AP
Probe
Target DNA
Membrane
Fig 4. Radioactive Western blot detected using PhosphorImager. Six
bands have been outlined for analysis using ImageQuant Auto Tracer
tool. The magnifier tool provides a closer view of the first band. The
Volume Report shows the results for the six objects.
ECF Western Blotting Kit offers alkaline phosphatasebased detection of protein blots. Fluorescein-labelled
anti-species secondary antibodies can be detected
directly and quantified by fluorescence imaging
instruments such as Typhoon, Storm, FluorImager
and ImageMaster VDS-CL. When amplification is
required, an anti-fluorescein alkaline phosphatase
conjugated tertiary antibody and ECF substrate are
used to catalyse the formation of stable fluorophores
that can be detected and quantified with a sensitivity
equivalent to ECL-based detection systems.
Following ECL detection, complete removal of primary and secondary antibodies from the membrane
without damaging the antigen allows multiple reprobing of membrane to either clarify or confirm results
when small or valuable samples are being analysed (1).
This compares favourably with radiolabels where ??emissions from 125I cause radiation damage to antibody activity and the label can detach from the
antibody, meaning reprobing is not recommended.
The protocols for radioactive antibodies are simple,
robust and generate durable hard-copy results on film
or storage phosphor screens for subsequent quantification and/or analysis. However, long exposures are
usually required, shelf-life is limited, and careful
monitoring and regulated disposal are essential when
handling radioactive isotopes. ECL, ECL Plus and ECF
provide non-radioactive alternatives offering antibody
economy due to improved sensitivity, and faster blot
analysis due to enhanced light emitting pathways that
generate quantifiable signal in minutes rather than
days. Chemiluminescent and chemifluorescent labelled
antibodies can be used for reprobing while radioactive
probes are preferred for multiple exposures.
ECL Plus is based on the enzymatic generation of an
acridium ester, which produces a more intense light
than standard ECL-based systems and has an emission
of longer duration. ECL Plus Western Blotting Detection System provides 10–20 fold improvement in
sensitivity over an equivalent ECL Western blotting
result and successful exposures can be made up to 24 h
after initiation of the detection reaction (Fig 5). The
increased sensitivity of ECL Plus means lower antibody concentrations and sample volumes can be
used ensuring the economical use of primary
antibodies and target proteins.
The generation of a fluorescent intermediate in the light
producing reaction pathway of ECL Plus means signal
can also be monitored on fluorescence imaging systems
such as Storm and FluorImager (2). ECL Plus is novel
in this ability as none of the luminol-based systems have
fluorescent characteristics. The substrate therefore
provides excellent sensitivity with the versatility to
allow use of the same Western blot for film exposure
and chemiluminescence or fluorescence instrument
scanning for quantification and rapid blot analysis.
References
1. Kaufmann, S.H., et al., Anal. Biochem. 161, 89–95,
(1987).
2. Amersham Pharmacia Biotech, Tech Tips. 173, (1997).
Fig 3. Outline of protocol for direct labelling method.
Life Science News 6, 2000 Amersham Pharmacia Biotech
Life Science News 6, 2000 Amersham Pharmacia Biotech
24
25
M
I
N
N
O
V
A
Substrate
T
I
O
S
F
O
R
U
M
I
N
N
O
V
A
T
I
O
platforms incorporating highly sensitive optic systems
such Typhoon 8600 and ImageMaster VDS-CL enable
the direct detection of chemiluminescence without the
need for intermediate exposures to films or screens.
However, unlike the signal produced by radioactive
probes, chemiluminescence is only linear over a narrow range and therefore offers limited quantification.
Light
Enzyme
Antibody
N
A
S
B
F
O
R
U
Fig 5. Target: mouse IgG slot blotted on to
PVDF (Hybond-P, RPN2020F):loadings:
doubling dilutions starting at 5 ng, detection: 1:2500 dilution of anti-mouse Ig HRP
conjugate (NA931) using (A) ECL Detection
Reagents (RPN2106) and (B) ECL Plus
Detection Reagents (RPN2132); exposure:on Hyperfilm ECL (RPN2103)
for 5 minutes.
Use of radioactivity in protein applications
Fluorescein
Probe
For detection of enzyme-labelled antibodies, the most
commonly used radiolabels are 125I and 35S, although
the range of radiolabelled antibodies is distinctly biased
towards 125I. This is largely due to the extensive use of
125I-labelled antibodies in clinical radioimmunoassays,
making them very accessible to researchers performing
Western blots. Furthermore, the signal produced by
125I and 35S is unaffected by enzyme, metal salts,
pH, or temperature, making them very useful for
new and uncharacterized systems often used during
life science research.
Target DNA
Membrane
Fig 2. Outline of indirect labelling method.
The fluorophores of hybridized probes can then be
visualized directly using systems such as Typhoon,
Storm, FluorImager, and ImageMaster VDS-CL.
Because different fluorophores are detected as different colours, it is possible to visualize two or more
different probes at one time, making fluorescence
multiplexing possible.
The signal produced by radiolabelled antibodies is
typically captured on autoradiographic film, providing
a permanent, hard-copy result. Multiple copies can be
produced from the same blot and results are easily
quantifiable by densitometry. However, filmless
autoradiography of radioactive Western blots is
possible through the use of re-usable, general-purpose
storage phosphor screens incorporated into image
analysis systems such as PhosphorImager™, Storm,
and Typhoon (Fig 4). These screens retain energy from
beta particles, X-rays and gamma rays and typically
50–90% less exposure time is required compared with
an equivalent exposure to conventional film.
With direct chemifluorescence labelling systems such
as AlkPhos Direct, AP is directly crosslinked to the
nucleic acid probe in a simple, 30 min reaction (Fig 3).
The probe is hybridized to the blot and incubated.
Detection is possible 1 h after hybridization. Direct
labelling saves 3–4 h compared to indirect labelling
methods (Fig 2). This is because the antibody conjugate incubation and associated blocking and washing
steps have been eliminated.
Results from fluorescent and chemifluorescent methods can be quantified using various imaging systems,
which allow filmless detection and subsequent image
analysis using powerful software packages including
ImageMaster™ and ImageQuant™.
Use of non-radioactive methods in
protein applications
Chemiluminescent detection systems were originally
designed for use with autoradiography film because
of the low levels of light produced. However, imaging
CDP-Star
or ECF substrate
N
Chemiluminescent and chemifluorescent methods for
the detection of enzyme-labelled antibodies have
largely replaced radioactivity in the immunodetection
of proteins. This is because non-radioactive systems are
highly sensitive, yield hard-copy results for subsequent
analysis after as little as 1min exposure to autoradiography film, and are safer to handle than radioisotopes.
Signal
ECL Western Blotting Analysis System is capable of
detecting < 1 pg of antigen on Hybond™ ECL membrane
making it suitable for routine Western blotting applications. Furthermore, the linear response exhibited by
Hyperfilm™ ECL to the signal means accurate quantification of proteins on ECL Western blots is possible.
AP
Probe
Target DNA
Membrane
Fig 4. Radioactive Western blot detected using PhosphorImager. Six
bands have been outlined for analysis using ImageQuant Auto Tracer
tool. The magnifier tool provides a closer view of the first band. The
Volume Report shows the results for the six objects.
ECF Western Blotting Kit offers alkaline phosphatasebased detection of protein blots. Fluorescein-labelled
anti-species secondary antibodies can be detected
directly and quantified by fluorescence imaging
instruments such as Typhoon, Storm, FluorImager
and ImageMaster VDS-CL. When amplification is
required, an anti-fluorescein alkaline phosphatase
conjugated tertiary antibody and ECF substrate are
used to catalyse the formation of stable fluorophores
that can be detected and quantified with a sensitivity
equivalent to ECL-based detection systems.
Following ECL detection, complete removal of primary and secondary antibodies from the membrane
without damaging the antigen allows multiple reprobing of membrane to either clarify or confirm results
when small or valuable samples are being analysed (1).
This compares favourably with radiolabels where ??emissions from 125I cause radiation damage to antibody activity and the label can detach from the
antibody, meaning reprobing is not recommended.
The protocols for radioactive antibodies are simple,
robust and generate durable hard-copy results on film
or storage phosphor screens for subsequent quantification and/or analysis. However, long exposures are
usually required, shelf-life is limited, and careful
monitoring and regulated disposal are essential when
handling radioactive isotopes. ECL, ECL Plus and ECF
provide non-radioactive alternatives offering antibody
economy due to improved sensitivity, and faster blot
analysis due to enhanced light emitting pathways that
generate quantifiable signal in minutes rather than
days. Chemiluminescent and chemifluorescent labelled
antibodies can be used for reprobing while radioactive
probes are preferred for multiple exposures.
ECL Plus is based on the enzymatic generation of an
acridium ester, which produces a more intense light
than standard ECL-based systems and has an emission
of longer duration. ECL Plus Western Blotting Detection System provides 10–20 fold improvement in
sensitivity over an equivalent ECL Western blotting
result and successful exposures can be made up to 24 h
after initiation of the detection reaction (Fig 5). The
increased sensitivity of ECL Plus means lower antibody concentrations and sample volumes can be
used ensuring the economical use of primary
antibodies and target proteins.
The generation of a fluorescent intermediate in the light
producing reaction pathway of ECL Plus means signal
can also be monitored on fluorescence imaging systems
such as Storm and FluorImager (2). ECL Plus is novel
in this ability as none of the luminol-based systems have
fluorescent characteristics. The substrate therefore
provides excellent sensitivity with the versatility to
allow use of the same Western blot for film exposure
and chemiluminescence or fluorescence instrument
scanning for quantification and rapid blot analysis.
References
1. Kaufmann, S.H., et al., Anal. Biochem. 161, 89–95,
(1987).
2. Amersham Pharmacia Biotech, Tech Tips. 173, (1997).
Fig 3. Outline of protocol for direct labelling method.
Life Science News 6, 2000 Amersham Pharmacia Biotech
Life Science News 6, 2000 Amersham Pharmacia Biotech
24
25
M