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Quantitative Western blottingImproving your data quality and reproducibility

March 3, 2015

Sponsored by:

Webinar Series

Participating ExpertsBrought to you by the Science/AAAS Custom Publishing Office Tibor Harkany, Ph.D.

Medical University of ViennaVienna, Austria

Åsa Hagner-McWhirter, Ph.D.GE Healthcare Life Sciences Uppsala, Sweden

Quantitative Western blottingImproving your data quality and reproducibility

March 3, 2015

Webinar Series

Sponsored by:

Quantitative Western blotting:tale of a neurobiologist end-user

Tibor Harkany

Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Austria

Department of Medical Biochemistry and Biophysics,Karolinska Institutet, Stockholm, Sweden

The “yes-no” phenomenon: detection without quantification(cell lines, receptors, signaling, genotypes)

ECL detection/film

50 kDa

cont

rol

enric

hmen

t

Differential localization Signaling/recruitment

Kilander et al, FASEB J (2014)Berghuis et al, Science (2007)

The dilemma of experimentalists: are my samples comparable?(cell lines vs. primary cells, cell cycle)

Cell line preparations:

• Large amount of sample available,• “Uniformity” through standardized transfection/manipulation, high(-er) efficacy• “Uniformity” through cell cycle and/or metabolic control (e.g. serum starvation),• High probability of survival, possible to propagate,• High signal-to-noise ratio.

Primary cell preparations:

• Small amount of sample available,• Transfection efficacy is generally low,• Cultured cell population is heterogeneous (cell cycle, metabolic status,

differentiation, various co-existent cell types from primary tissues),• Propagation is not possible (“one-off samples”),• Ectopic expression of various non-target proteins (result confounds),• Low signal-to-noise ratio.

Western analysis on limited and metabolically active cells(neurons, low signal-to-noise)

EXAMPLE - ECF detection

50,000 neurons 1,000,000 PC12 cells(selective isolation) (serum starved)

Berghuis et al, PNAS (2005)

The dilemma of experimentalists: how to standardize?(differentiation, cell cycle)

EXAMPLE 1 - Differentiation

Control culture(e.g. neurons)

Differentiationfactor

Differentiated culture(neurite outgrowth)

• Experimental manipulation affects cellular morphology (e.g. processes), anddifferentiation state (= differentiation factor-driven proteome),

• Cytoskeletal proteins (most often used as “loading controls”, tubulin, actin) aredifferentially expressed, thus their use could bias experimental results,

• Gapdh and other “house-keeping” proteins are affected by neuronal differentiation,limiting their use for comparisons at different developmental time points,

• Cell numbers might be (or not) affected by differential survival.

EXAMPLE 1 – Differentiation(time course analysis in fetal/neonatal brain)

Keimpema et al, J Neurosci (2010)

Time course analysis during brain development(normalized expression)

A A1 A2

The dilemma of experimentalists: how to standardize?(differentiation, cell cycle)

EXAMPLE 2 – Cell cycle control, proliferation

Control culture(e.g. stem cells)

• Experimental manipulation affects cell cycle,• Differentiation alters cell state, rendering the culture more asynchronous and

difficult to control,• Alternatively, cell proliferation can affect the amount of cells and bias for

individual changes (“dilution effects” for subpopulations of cells ifheterogeneity exists).

Differentiation

Proliferation

The dilemma of experimentalists: how

to standardize?(differentiation, cell cycle)

The dilemma of experimentalists: several ways to the “same” result?(time-course experiments)

A (single control at longest time point) B (internal control for each time point)

Keimpema et al, PNAS (2013)

Standardization for molecular pharmacology(differentiation, cell cycle)

β-III-tubulinVAChT

DUAL COLOR imaging for better correlation of internal standards (infrared dye imaging)

Benefits:• Direct correlation between loading marker and target,• Extended linearity of the signals,• Controlled for saturation.(Drawback: membranes cannot be stripped/re-probed)

Correlation of in vivo and in vitro data: focus on tissue complexity (signal dilution, signal interactions, “system noise”)

Correlation of Western and histochemical datasets(protein localization vs. protein levels)

Correlation of Western and histochemical datasets(protein localization vs. protein levels)

Controlling total protein load in complex tissues (Cy5/Cy3 imaging)

Developmental profiling – “unbiased” quantification

Total protein Target

• Cy5 labeling for total protein• Cy3 labeling for target protein

Developmental/regional expression

Conclusions(evolution of technology)

• Experimental paradigm defines the need of technical complexity,• Controls must be carefully selected and be based on all

experimental variables,• Comparative analyses require highest rigor,• The use of more than one method should be favored to address

scientific problems,• Detection of total protein load seems best suited for quantitative

Western blotting. The novel technology also adheres to recentpublishing guidelines, requiring the presentation of uncutmembranes (“transparent reviewing process”).

Acknowledgements

Paul BerghuisMarton B. DobszayHylke Jan KingmaJan MulderOrsolya K. PenzErik KeimpemaGiuseppe TortorielloKlaudia BarabasAlan AlparPhilip Cowie

Lauren SpenceRoman RomanovMingdong ZhangDaniela CalvigioniJanos Fuzik

novo nordisk fonden

Katarzyna MalenczykValentina CinquinaJoanne BakkerGloria Arque

Participating ExpertsBrought to you by the Science/AAAS Custom Publishing Office Tibor Harkany, Ph.D.

Medical University of ViennaVienna, Austria

Åsa Hagner-McWhirter, Ph.D.GE Healthcare Life Sciences Uppsala, Sweden

Quantitative Western blottingImproving your data quality and reproducibility

March 3, 2015

Webinar Series

Sponsored by:

Imagination at work.

Åsa Hagner McWhirter, Ph.D.March 3rd 2015

Quantitative Western blotting: Improving your data quality and reproducibility

Outline

Critical factors for quantitative Western analysis• Detection system: Chemiluminescence versus fluorescence• Normalization • Imaging and analysis• Reproducibility and standardization

20

Western detection methods

HRP conjugated secondary antibody

Dye conjugated secondary antibody

ECL detection reagent

Chemiluminescence

Fluorescence

Primary antibody

Electrophoresis&

Transfer

Imaging&

Analysis

Chemiluminescence Fluorescence

21

Chemiluminescence Western blotting

Indirect signal involving an enzymatic reactionDetection using film or CCD cameraSensitive (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

22

Fluorescence Western blotting

Direct signal with dye labeled secondary antibodiesDetection using laser or CCD imagerSensitive (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

23

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

24

Detection systemSignal stability is critical for accurate quantitation

1 hour

Sig

nal

Sig

nal

Em

ission filter

532 nm

Cy™3 Cy5

633 nm

Em

ission filter

• Dyes detected simultaneously

• Laser for excitation• Filter defines capture of

emitted signal• Spectrally well resolved dyes • Minimal cross-talk

500 600 700 nm

Detection systemFluorescence enables multiplex detection

25

- 5 15TGF-β stim. time: 30 60 120min

Akt

pAkt

- - - - - - - +LY (inhibitor):

Data courtesy of Dr. Marene Landström and Anders Marcusson, Ludwig Institute for Cancer Research, Uppsala, Sweden.

Cy™ 3

Cy 5

overlay

Two proteins of the same Mw can easily be detected, even when one is at significantly lower concentration

Detection of two targets of the same Mw

26

Normalization

27

Why is normalization required?

ERK1/2

GAPDH

28

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

Endogenous protein Total protein• 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

29

2.5µg – 20µg sample

Nor

mal

ized

ratio

Targ

et/C

ontro

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

Sign

al

Sample amount (µg)

ControlTarget

30

00.010.020.030.040.050.060.070.08

1 2 3 4 5 6 7 8 9 10

Validation of house-keeping proteins critical for accurate normalization results

31

0500

1000150020002500

0 2 4 6 8 10 12 14 16 18 20

0

2000

4000

6000

0 2 4 6 8 1012 141618 20

Tubulin

GAPDH

Total protein Actin

EGF stimulationA431 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

32

Cy5

tota

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

Image capture and analysis

33

Image capture

34

• Imager hardware requirements High sensitivity and wide dynamic range Minimum of cross-talk Optimal signal capture without saturation Resolution

• Detection reagents requirements Should match imager specifications

Image analysis

- Lane detection- Band detection/ target definition- Background subtraction

35

Strategy &Sample prep

Property of detection reagents

Normalization

High qualityimagingImage

analysis

Protocols & compatible products

Separationresolution

Every Western step is important for data quality

Transfer efficiency

36

Reproducibility

37

Challenges in traditional Western blotting

• Number of steps

• Non-standardized

• Skills required to obtain quantitative data

• Poor reproducibility between blots and labs

38

Standardization of Western workflowfor low variation between blots and operators

• Same equipment, settings and procedures- Electrophoresis - Transfer- Probing protocol- Image capture- Image analysis and evaluation

• Same consumables and detection reagents• Same handling and exposure times

39

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

40

SDS-PAGECy™5 pre-labeledsamples

Western blotCy5 and Cy3 labeledsecondary antibodies

41

User 1

User 2

User 3

Cy3 Non-normalizedtarget 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

00.005

0.010.015

0.020.025

0.030.035

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

00.010.020.030.040.050.06

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

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

Amersham, Cy and Cydye 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, Björkgatan30, 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.

© 2015 General Electric Company – All rights reserved.

First published March 2015.

GE Healthcare Bio-Sciences AB, a General Electric Company.

GE Healthcare Bio-Sciences AB

Björkgatan 30

751 84 Uppsala

Sweden

www.gelifesciences.com

Participating ExpertsBrought to you by the Science/AAAS Custom Publishing Office To submit your

questions, click theAsk a Question

button

Quantitative Western blottingImproving your data quality and reproducibility

March 3, 2015

Webinar Series

Sponsored by:

Tibor Harkany, Ph.D.Medical University of ViennaVienna, Austria

Åsa Hagner-McWhirter, Ph.D.GE Healthcare Life Sciences Uppsala, Sweden

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Quantitative Western blottingImproving your data quality and reproducibility

March 3, 2015

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