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.“

4

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


How is Western done?

Conventional manual Western set up

Integrated Amersham™ WB system set up

8

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 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


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

13

Fluorescence multiplexing

Protein normalization

Integrated scanner

• Designed for accurate quantitation

• Standardized for reproducible results across labs and users, through system integration


Stay tuned with your Western blotting run Amersham WB watch app for iOS and Android

14

•Monitor the experiment progress remotely

•Get notifications when time to intervene

•View resulting images instantly after Gel card or Membrane scan


Available soon...

15

Designed for reproducible data across users and labs

16

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 %

17

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

18

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%

19

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

20

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 softwareApplications supported

Non

-qu

an

tita

tive

Weste

rn.

SD

S-P

AG

E f

or

an

aly

sis

of

imp

uri

ties.

SD

S-P

AG

E f

or

targ

et

pro

tein

exp

ressio

n a

naly

sis

.

Weste

rn f

or

an

aly

sis

of

mod

ificati

on

s.

Weste

rn f

or

qu

an

tita

tive

com

pari

son

betw

een

com

ple

x

bio

. sam

ple

s.

Weste

rn f

or

iden

tity

con

firm

ati

on

.

Easy WesternNon-quanititive western and western for identity confirmation

24

•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

25

•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

26

•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

27

•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

28

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


Bax/Actin

Western with total protein normalization Bax levels in UV treated HeLa cells

29

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.


Bax

1 2 3 4


Bax/total protein

Easy Western - Tissue screening

30

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.


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

13

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


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


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.


Learn more about Amersham WB system






GE, GE monogram and imagination at work are trademarks of General Electric Company.

Amersham, ÄKTA, Superdex, Sepharose and Cy are trademarks of General Electric Company or one of its subsidiaries.

CyDye: This product is manufactured under an exclusive license from Carnegie Mellon University and is covered by US patent numbers 5,569,587 and 5,627,027.

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.

A license to use the CyDye products for commercial purposes is subject to a separate license agreement with GE Healthcare.

Commercial use shall include:

1. Sale, lease, license or other transfer of the material or any material derived or produced from it.

2. Sale, lease, license or other grant of rights to use this material or any material derived or produced from it.

3. Use of this material to perform services for a fee for third parties, including contract research and drug screening.

If you require a commercial license to use this material and do not have one, return this material unopened to GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden and any money paid for the material will be refunded.

All third party trademarks are the property of their respective owners.

All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.

© 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

amersham western blotting system

Science

amersham wb systemsetting

amersham wb way

published wb data

time amersham wb systemwe

reliable data western

amersham imager

integrated western blot

western blottingimagination