fluorescence detection & critical factors for quantitative western blotting
TRANSCRIPT
Imagination at work.
February 2015
Fluorescence detection and critical factors for quantitative Westerns
Content
• Western blotting - workflow and results
• Chemiluminescence versus Fluorescence
• Critical factors for quantitative Western analysis
• How Amersham™ WB system is designed to maximize
data quality
2
1. Protein separation by SDS-PAGE
2. Protein transfer to a membrane
3. Blocking and probing with primary
antibody specific to target protein
4. Probing with a labeled secondary
antibody specific to primary antibody
5. Imaging using film, CCD camera
or laser scanner
6. Image analysis (dedicated software)
Western blotting workflow
1
2
3
4
3
What does a Western blot deliver?
Western blots are widely used in many types of
applications for confirmatory or quantitative
analysis of specific target proteins
• Protein band signal confirms protein presence and
identity
• Position of band informs about protein molecular
weight, confirm expected size
• Intensity of band informs about protein amount in
the sample4
Challenges in traditional Western blotting
• Number of steps and time consuming
• Different protocols and ways of working
• Difficult to obtain quantitative data
• Poor reproducibility
5
Western detection methods
HRP Secondary
antibody
Cy™ Dye
Secondary
antibody
Amersham™ ECL
detection reagent
Chemiluminescenc
e
Fluorescence
Primary antibody
Electrophoresis
&
Transfer
Imaging
&
Analysis
Chemiluminescence Fluorescence
6
Chemiluminescence Western blotting most suited for confirmatory analysis
Indirect signal involving an enzymatic reaction
Detection using film or CCD camera
Sensitive (low pg)
Medium dynamic range (~2 orders with CCD)
Pros
+ Low abundant proteins detection
Cons
- Unstable signals, variation between blots
- Single protein detection. Stripping and reprobing required
for second protein detection
- Requires knowledge, skills and controlled ways of working
to be quantitative
7
Fluorescence Western blotting most suited to quantitative analysis
Direct signal with dye labeled secondary antibodies
Detection using laser or CCD imager
Sensitive (pg)
Broad dynamic range (~3 orders)
Pros
+ Multiplex detection possible
+ Reliable normalization simultaneously on same blot
+ Stable signals, reproducible between blots
Cons
- Care needed to avoid fluorescence contamination
- Sometimes requires higher concentration of
primary antibody
8
What is critical for quantitative analysis?
• Detection system
• Normalization
• Image capture and analysis
• Reproducibility and standardization
9
Chemiluminescence• Unstable signal declining within minutes
• High variation between blots
• Skills and controlled handling needed
for accurate quantitation
• Good choice for confirmatory Westerns
Fluorescence• Stable signal for months
• High reproducibility
• Accurate quantitation
• First choice for quantitative Westerns
3 months
10
Detection systemSignal stability is critical for accurate quantitation
3 months1 hour
Sig
na
l
Sig
na
l
Detection systemFluorescence
• Sensitive (pg levels)
• Broad dynamic range (~3 orders of magnitude)
• High signal to noise ratio
• Reliable normalization without strip and reprobing
Filte
r for 5
70
nm
em
issio
n
532 nm
Cy™3 Cy5
633 nm
Filte
r for 6
70
nm
em
issio
n
• Dyes detected simultaneously
• Precise excitation light from laser or LED epi sources
• Filter defines capture of emitted signal
• Spectrally well resolved dyes
• Minimal cross-talk
500 600 700nm
Detection systemFluorescence enables multiplex detection
1
2
Cy5Cy™3 Cy3/Cy5
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Detection systemAccurate detection of 2 targets of same Mw by multiplex
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Experiment:
ERK phosphorylation upon UV treatment of HeLa cells. The MW shift is very low upon phosphorylation
and cannot typically be distinguished using chemiluminescence. Both phosphrylated and non-
phosphorylted ERK can easily be identified and reliably quantitated in the same sample using Cy3 and
Cy5 labelled secondary Ab’s.
Why is normalization required?
1
5
ERK1/2
GAPDH
1 2 3 4 5 6 7 8
To correct for uneven loading between wells due to:
• Protein quantitation errors
• Errors in cell number estimation e.g. one culture dish per sample
• Pipetting errors
Loading controls for normalization
16
• GAPDH, tubulin and actin are most
commonly used
• May have limited detection range
• Single protein signal
• May be affected by cellular treatments
• Antibody optimization needed
• Well-known and widely used method
• Total protein signal from Cy™5 pre-
labeled proteins or protein stains
• Broad detection range
• Sum of many protein signals
• Minimally or not affected by cellular
treatments
• Antibody independent
• More recently introduced method
Total proteinEndogenous protein
2.5µg – 20µg sample
Norm
aliz
ed r
atio
Ta
rge
t/C
on
tro
lValidate your normalization method
Normalization method requirements:
• Proportional response of target
and control signals
• The same sample should give the
same T/C ratio regardless of
sample load
0
500
1000
1500
2000
2500
3000
3500
0
10000
20000
30000
40000
50000
60000
0 5 10 15 20 25
tot sp 20x
ERK 2
Sig
nal
Sample amount (µg)
Control
Target
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0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
1 2 3 4 5 6 7 8 9 10
Validation of house-keeping proteins critical for accurate normalization results
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0
500
1000
1500
2000
2500
0 2 4 6 8 10 12 14 16 18 20
0
2000
4000
6000
0 2 4 6 8 10 12 14 16 18 20
Tubulin
GAPDH
Total protein Actin
EGF stimulation
A431 cell lysate
Actin
• Select house-keeping protein and
probing conditions (antibody dilution)
producing proportional response in the
sample range to be used.
• Make sure the house-keeping
protein is not affected by treatment
HK
pro
tein
sig
nal
CHO cell lysate (µg)
Normalization using total protein
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Cy5 t
ota
l pro
tein
sig
nal
Sample amount (µg)
0
5000
10000
15000
20000
25000
30000
0 5 10 15 20 25
Reliable normalization method:
• Antibody independent
• Not affected by treatments
• Sum of many protein signals
• The whole lane or part of the lane
can be used
Cy™5 total protein pre-labeling
Quantitative image captureImager requirements
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• High sensitivity and wide dynamic range
• Minimal cross-talk between channels
• Optimal signal capture without saturation
• Optimal resolution
• Detection reagents to match imager specifications
Quantitative image analysis
Image analysis software
requirements:
- Optimal lane detection
- Optimal band detection/Target definition
- Optimal background subtraction
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Strategy &
Sample
prep
Property of
detection
reagents
Normalization
High
quality
Imaging
Image
analysis sw
Protocols
& compatible
products
Separation
resolution
Every Western step is critical for data quality
Low background
High signal to noise
Optimal signal capture
Sensitivity
High signal to noise
Broad dynamic range
Reproducibility
Optimal signal definition &
background removal
for accurate quantitationRemove technical
variation
Reproducibility
Sensitivity
High signal to noise
Broad dynamic range
Sharp bands for
optimal target definition
Maximize target
abundance in sample
Transfer
efficiency Sensitivity
24
Challenges in traditional Western blotting
• Number of steps
• Different protocols and ways of working
• Difficult to obtain quantitative data
• Poor reproducibility between blots and labs
Amersham™ WB system
• Integrated instrument for
electrophoresis, transfer, probing,
scanning and image analysis
• Laser scanner
• Total protein normalization
• Standardized workflow,
reproducible results
• Easy to use, error proofed in
majority of steps
25
SDS-PAGE
Cy™5 pre-labeled
samples
Western blot
Cy5 and Cy3 labeled
secondary antibodies
High reproducibility across experiments and operators with Amersham™ WB system
• Standardized controlled protocols with optimized settings for
- Electrophoresis (with automated stop by front detection)
- Transfer
- Automated probing protocol
- Scanning with automated pre-scan
- Automated image analysis and evaluation
• Optimized consumables and detection reagents
• Error proofed in majority of steps
• Default or manual settings in every step for a controlled process
26Typical CVs 5-10% or less
27
User 1
User 2
User 3
Cy3 Non-normalized
target signals (CV%)Image overlay
Cy3/Cy5
Normalized target
signals (CV %)
0
1000
2000
3000
4000
1 2 3 4 5 6 7 8 9 101112
0
1000
2000
3000
4000
1 2 3 4 5 6 7 8 9 101112
6.3
9.8
0
2000
4000
6000
1 2 3 4 5 6 7 8 9 101112
8.2
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
1 2 3 4 5 6 7 8 9 10 11 12
0
0.02
0.04
0.06
0.08
1 2 3 4 5 6 7 8 9 10 11 12
0
0.01
0.02
0.03
0.04
0.05
0.06
1 2 3 4 5 6 7 8 9 10 11 12
3.5
4.6
5.0
Reproducible results with the Amersham™ WB
system
Amersham™ WB systemSetting a new standard in Western blotting
Integration of the workflow and optimization of
protocols and components in Amersham™ WB
system are designed for:
High reproducibility across experiments
and operators
Quantitative data you can trust every
sample, every time
28
Learn more at
http://www.gelifesciences.com/artofwesternblotting
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First published February 2015.
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