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

13

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.

Normalization

Quality data from every Western blot

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

17

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

18

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

19

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

Image capture and analysis

20

Quantitative image captureImager requirements

21

• 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

22

Reproducibility

23

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

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, 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.

© 2015 General Electric Company – All rights reserved.

First published February 2015.

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

GE Healthcare Bio-Sciences AB

Björkgatan 30

751 84 Uppsala

Sweden

www.gelifesciences.com

30