Download - Amersham Western Blotting System
Imagination at work.
http://www.gelifesciences.com/artofwesternblotting
Amersham™ WB systemThe new standard in Western blotting
It is time to change the face of Western
Expressed by researchers, the reality of Western blotting• WB lacks standardization and
quantitation
• WB requires extensive repeat runs and control experiments to achieve reliable data
• Reliable data is key
Hence, we designed Amersham™ WB system in dialogue with scientists to deliver
• reproducible data
• quantitative data
• less repeats & control experiments
designed to minimize variability every sample, every run. Amersham™ WB system
The voice of scientists across the globeReactions after seeing Amersham WB system
‘This is attractive… if blots could be normalized using standard
protocols’
‘Quantitation made easy’
‘You just can´t get it wrong’
‘Easy to learn’
‘Improved reproducibility…’
‘… enable scientists to compare and
repeat published WB data.’
‘Integration of 1D & WB is where I see your most important features
‘
‘With today´s hands-on solutions for WB, each new employee will waste a month´s worth of results
before getting it right‘
‘… good quantitation, is critical to get it right…’
‘This is what we all want.’
Designed to provide consistent Western blot
data, every sample, every time
Amersham™ WB system
We set out to change the face of Western
“Scientists need to get faster to Western blotting results that they can trust to move forward their research.
We have the capabilities required to design an integrated Western blot system that will minimize assay variability to repeatedly and reliably provide quantifiable data.
By combining data normalization with a standardized and monitored process, we will allow scientists to retain full control over their experiments and get more out of each blot.“
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Anders Fält, R&D Director GE Healthcare
Background: Western blotting a widely used analytical technique
Sample prep
Gel Electrophor
esis
Transfer
Antibody probing
Detection
Imaging
Analysis
Widely applied:• Used in ~37.000 labs• 60% of publications in leading scientific
journals contain WB data
Valued for its benefits:• Well-established & trusted• Available in most labs• Ability to identify and quantify a specific
protein in a complex mixture, e.g. a cell extract
Accepted with its challenges:• Lack of standardization and quantitation• Requires extensive repeat runs and
controls to achieve reliable data
Western Blotting – The Amersham WB way
Fully integrated Amersham WB system
Designed to minimize assay variability to deliver quantifiable data, every sample, every time.
Sample prep
Gel Electrophor
esis
Transfer
Antibody probing
Detection
Imaging
Analysis
Amersham™ WB system
How is Western done?
Conventional manual Western set up
Integrated Amersham™ WB system set up
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Amersham™ WB systemSetting the new standard in Western blotting
Standardization of workflow & integration of components
Amersham WB system is designed for:
• High reproducibility across experiments & operators
• Quantitative data you can trust, every sample, every time.
Amersham™ WB system
Amersham WB systemKey components of the system
Transfer and probing unit
+
PVDF card
Electrophoresis and laser scan unit
Transfer sandwich
Gel card
Software for: • Experimental set-
up• Progress monitoring• Experiment
evaluation
Amersham™ WB system
Reproducibility - across experiments and operatorsAmersham WB system is a fully integrated system for separation, transfer, detection, and quantitative analysis of proteins that is designed to provide consistent, quantitative data for every sample, every time.
Standardized, true Western - High conformity w. previous
Western data
Consumables integration – Gel cards & PVDF
cards• Optimized Amersham™ WB
Gel card and Amersham WB PVDF card.
• Keyed to fit unambigously in the instrument and in Amersham WB transfer holder.
• Data matrix tag identification of Gel cards and membranes at critcal steps in the workflow to secure link between sample and result.
• All critical processing parameters have been standardized and optimized for best possible result:
- Protocols - Volumes - Durations - Evaluation settings
• Automated processing ensures high reproducibility
Amersham™ WB integrated software for the entire
workflow• Experiment and sample:- Define the experiment- Add information: samples and antibodies
• Transfer:- Fine tune running conditions.
- Monitor the experiment• Evaluation:- Confirm results- Fine tune evaluation parameters. - Extract information: documentation and publication
Quantitative data you can trustHigh quality of data by combining three elements: a system design that standardizes and monitors the process; optimized consumables and protocols that minimize assay variability; and built-in data normalization.
Quantitative fluorescent labeling – High sensitivity and wide linear dynamic
range
Quantitative laser scanner detection –
Chosen for its superior performance
• Laser point scanner technology was chosen for its superior sensitivity and wide dynamic range.
• Two separate scan heads with individually optimized optics for detection of the two different dyes (Cy™3 and Cy5) with a minimum of cross-talk.
• Amersham™ WB Cy™5 Dye reagent used for total protein pre-labeling.
• Amersham Cy3 and Amersham Cy5 pre-labeled secondary antibodies used for Western detection.
Fluorescence multiplexing for total protein normalization
For accurate quantitation, built-in
protein normalization corrects for
uneven sample loading between
wells due to:• Protein quantitation errors• Estimate sample amounts
e.g. one culture dish per sample
• Pipetting errors
System integration
Consumables integration
Software integration
Amersham WB system
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Fluorescence multiplexing
Protein normalization
Integrated scanner
• Designed for accurate quantitation
• Standardized for reproducible results across labs and users, through system integration
Amersham™ WB system
Stay tuned with your Western blotting run Amersham WB watch app for iOS and Android
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•Monitor the experiment progress remotely
•Get notifications when time to intervene
•View resulting images instantly after Gel card or Membrane scan
Amersham™ WB system
Available soon...
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Designed for reproducible data across users and labs
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Reproducible Cy™5 pre-labeling of proteins
Highly reproducible protein labeling protocol
225 kDa
97 kDa
66 kDa50 kDa
35 kDa
25 kDa20 kDa
14 kDa10 kDa
14 individual samples containing a mix af Conalbumin and Aldolase (0.1µg/µl) were pre-labeled with Cy5 and applied to an Amersham™ WB gel card.
CV(%) of the signal intensity and ratio between the 2 proteins were calculated for each reaction.
ConalbuminCV: 4.3 %
AldolaseCV: 7 %
Ratio Conalbumin/AldolaseCV: 5.7 %
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Low inter-assay variability
Without normalization, the CV of target protein volumes for a single loading amount within a membrane is 6% and between membranes 8% on average
Cy™3 tubulin Operator Membrane 1
(% CV)
Membrane 2
(% CV)
Membrane 3
(% CV)
Membrane 1-3(% CV)
4 ug
User 1 1,4 6,4 7,3 12,2User 2 12,1 7,8 5,8 8,8User 3 5,9 3,9 4,8 5,0
12 ug
User 1 1,7 10,4 7,1 7,5User 2 3,9 7,0 10,6 9,4User 3 7,4 6,7 3,4 9,6
20 ug
User 1 2,0 9,8 6,4 6,4User 2 7,5 4,2 7,7 7,6User 3 3,2 5,5 7,3 7,3
Quadruplicate samples: 4, 12 and 20 µg CHO cell lysate3 membranes3 users
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Low intra-assay variability Even with major loading variation
Normalized values within a membrane are on average 8% for the whole 4-20µg loading range
Ratio Cy3 tubulin and Cy5 total protein loading range 4-20µg
Operator Membrane 1
(% CV)
Membrane 2
(% CV)
Membrane 3
(% CV)User 1 10,0 8,7 7,5
User 2 8,3 5,6 5,9
User 3 8,8 9,7 7,2
Cy™3 tubulin Cy5 total protein
Quadruplicate samples: 4, 12 and 20 µg CHO cell lysate3 membranes3 users
Quick learning curve for first time users
First time users achieve normalized %CV values below 5%
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CV 5.0%User 1
CV 4.6%User 2
User 3 CV 4.5%
CV 3.5%User 412 x 7.5µg CHO cell lysateCy3 tubulin or ERK ½Cy5 total protein
Cy™3/Cy5 normalized ratio
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Uniform results across usersIrrespective of experience
Experienced & first time users achieve similar CVs
Cy™3 tubulin target signals variability (CV%)
First time users Experienced users
9,3 8,4
Two experienced and two first time users running 12 CHO cell lysate samples each (12 µg and 7,5 µg, respectively)
Applications supported
Amersham WB systemTypical applications supported
Non-quantitative WesternLooking for ”Yes/No” answer to questions like:- Does this expression system work?
Confirmatory WesternCharacterization of target protein products by confirming protein identity
Semi-quantitative comparison of samplesDifferent biological states, effect of stimulation, treatment, knock-outs, mutations, time progression etc.
Quantitative WesternQuantitative comparison of samples representing different biological state
Electrophoresis Western Blotting
Fraction analysisRefine your fractionation and pooling strategy Good fit with ÄKTA™
Purity analysisUse the wide dynamic range to relate intensities for different bands to the target protein
Absolute protein quantitationUsing a standard curve
Amersham™ WB system
Amersham™ WB softwareApplications supported
Non
-qu
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Weste
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.
Easy WesternNon-quanititive western and western for identity confirmation
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•Quick confirmation of protein identity and easy sample-to-sample comparison.
•One primary antibody against the specific target.
•A corresponding, labeled secondary antibody for the detection.
•Optionally two sets of antibodies for detecting two targets separately.
Western with endogenous protein normalization
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•Quantitative comparison of a target protein between samples.
•Two primary antibodies, of different species, against two different targets.
•Two corresponding, differently labeled secondary antibodies for the detection.
•One of the targets is used as control for normalization.
Western with total protein normalization
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•Quantitative comparison of a target protein between samples.
•A primary antibody against the specific target.
•A corresponding, labeled secondary antibody for the detection.
•Pre-labeled total protein is detected separately in the membrane and used as control for total protein normalization.
Easy SDS-PAGE
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•Comparison within and between samples.
•This experiment uses only the electrophoresis part of the workflow.
•Pre-labeled total protein is detected in the Gel card immediately after electrophoresis.
•Samples need to be pre-labeled for the detection.
AmershamTM WB system
Western with endogenous protein normalization Bax levels in UV treated HeLa cells
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Is Bax expression affected by UV treatment?Sample 20 µg Hela lysate control/UV treated1. 100% C (lanes 3-4)2. 67% C, 33% T (lanes 5-6)3. 33% C, 67% T (lanes 7-8)4. 100% T (lanes 9-10)
Gel 8-18 % gradientPrimary AbRabbit anti-bax 1:000Mouse anti-actin 1:2500Secondary AbCy™5 labeled anti-rabbit 1:2500 Cy3 labeled anti-mouse 1:2500
Result/ConclusionBax was found to be downregulated upon treatment with UV when signals were normalised against house-keeping protein.
Actin
Bax
1 2 3 4
AmershamTM WB system
Bax/Actin
Western with total protein normalization Bax levels in UV treated HeLa cells
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Is Bax expression affected by UV treatment?Sample Cy™5 pre-labeled20 µg Hela lysate control/UV treated1. 100% C (lanes 3-4)2. 67% C, 33% T (lanes 5-6)3. 33% C, 67% T (lanes 7-8)4. 100% T (lanes 9-10)
Gel 8-18 % gradientPrimary AbRabbit anti-bax 1:1000Secondary AbCy3 labeled anti-mouse 1:2500
Result/ConclusionBax was found to be downregulated upon treatment with UV when signals were normalised against Cy5 total protein.
AmershamTM WB system
Bax
1 2 3 4
AmershamTM WB system
Bax/total protein
Easy Western - Tissue screening
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Expression levels and patterns in different types of rat tissue?Sample 20µg rat tissueGel 8-18 % gradientPrimary Ab’sRabbit anti beta-catenin 1:750Mouse anti actin 1:750Secondary Ab’sCy™5 labeled anti-rabbit 1:2500 Cy3 labeled anti-mouse 1:2500
Result/Conclusion• Tissue type depedent expression
levels.• Target variants and different relative
amounts vary depending on tissue type.
AmershamTM WB system
Beta catenin(66 and 14 kDa)
Actin (42kDa)
Kidn
eyTe
stis
Actin
Beta-catenin
Degraded Beta-catenin?
Lung
Brai
n
225
9766503525201410
kDa
How much and how pure is the His tagged target in my fractions?
Sample Cy5 pre-labeled Sf-21 insect cell culture supernatant and purified sample fractionsGel 8-18 % gradientPrimary AbMouse anti-His Secondary AbCy3 labeled anti-mouse 1:2500
Result/Conclusion• The target protein was enriched.• Unspecific protein was washed
away.
Easy Western Purification of His tagged protein
His Mag Sepharose™ excel1 Mw2 SM3 FT4 wash 15 wash 26 wash 37 Elution 18 Elution 29 Mw
Buffer Exchange prior to Cy5 pre-labelingusing Amersham™ WB MiniTrap kit
% 4W1
5W2
6W3
7E1
8E2
Target 5 14 40 82
87
Impurity
95 86 60 18
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Cy™5 total protein
Target
1 2 3 4 5 6 7 8 9
Impurity
1 2 3 4 5 6 7 8 9
Cy3 target protein
Membrane images
AmershamTM WB system
Does UV stimulation induce phosphorylation of ERK?Sample Cell lysate (15µg) from HeLa cells control and UV treated 0 (1), 25 (2), 50 (3), 75 (4) and 100%(5) UV treated Gel 8-18 % gradientPrimary AbRabbit anti-ERK 1:5000Mouse anti-pERK 1:2000Secondary AbCy5 labeled anti-rabbit 1:2500 Cy3 labeled anti-mouse 1:2500
Result/Conclusion• ERK phosphorylation was
induced by UV treatment. • Two targets of same Mw
can be multiplexed.
control
AmershamTM WB system
Cy5Cy™3 Cy3/Cy51 2 3 4 5 1 2 3 4 5 1 2 3 4 5
pERK ERK pERK/ERK
Western with two targets of same Mw ERK phosphorylation in HeLa cells
Product overview
Key components of Amersham WB system
Transfer and probing unit
+
PVDF card
Electrophoresis and laser scan unit
Transfer sandwich
Gel card
Software for: • Experimental set-
up• Progress monitoring• Experiment
evaluation
System integration, designed to minimize variability Quantifiable data, every sample, every time
Total protein pre-labeling protocol
Electrophoresis protocol
Westernprotocol
Electrophoresisreagents & consumables
Total proteinpre-labeling reagents
Westernreagents & consumables
Electrophoresisfunction
Transferfunction
Wash, incubation &drying functions
Detectionfunction
Detectionfunction
Add your sample and
primary antibody
Amersham WB systemSetting a new standard in Western blotting
Integration of the workflow and optimization of protocols and components in AmershamTM WB system are designed for:
High reproducibility across experiments and operators
Quantitative data you can trust every sample, every time.
Amersham™ WB system
Learn more about Amersham WB system
Amersham™ WB system
http://www.gelifesciences.com/artofwesternblotting
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Amersham, ÄKTA, Superdex, Sepharose and Cy are trademarks of General Electric Company or one of its subsidiaries.
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The purchase of CyDye products includes a limited license to use the CyDye products for internal research and development but not for any commercial purposes.
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© 2014 General Electric Company – All rights reserved.
First published June 2014.
GE Healthcare Bio-Sciences AB, a General Electric Company.
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
www.gelifesciences.com
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