Fluorescent Western Blotting

February 28, 2012

Dr. Martin Broadstock

Edmond and Lily Safra Research Fellow

King’s College London

Topics to be covered..

Introduction to fluorescent western blotting

• ECL versus fluorescence

• Key advantages

Application of fluorescent • Why use fluorescent westerns?

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fluorescent western blotting to research

• Challenges experienced in the use of fluorescent westerns

• Trends and the future of fluorescent westerns

Common pitfalls and technical challenges in fluorescent western blotting

• Blocking

• Autofluorescence

• Imaging systems

• Storage and handling of membranes

My current research

Investigates synaptic dysfunction in neurodegenerative diseases

• Western Blots (all fluorescent)

• Immunohistochemistry

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• Immunohistochemistry

• Immunocytochemistry

• In vivo studies

• Fluorescence is now the only western blotting detection system within the Wolfson Centre for Age-Related Diseases at King’s College London Broadstock M et al. Synaptic protein expression is

regulated by a pro-oxidant diet in APPxPS1 mice. J Neural Trans 119, 493-496 (2012)

Comparison of ECL with fluorescence detection

Loading & running gel

Transfer gel to membrane

Blocking

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Primary antibody staining

Secondary antibody staining

ECL detection reagents Analyze imageExpose to film

HRP Fluorescence

Advantages of fluorescent detection

Improved Signal

Problems Advantages

Unstable signal with ECL Fluorescent signals are stable for at least one year

Obscuring smaller weight proteins Fluorescence allows smaller weight proteins to be more easily resolved

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proteins to be more easily resolved

Reduced sensitivity of ECL Fluorescence generally has increased sensitivity when compared with ECL(Gingrich, JC et al. Multiplex detection and quantitation of proteins on western blots using fluorescent probes. Biotechniques 29, 636-642 (2000)

Advantages of fluorescent detection

Reduced cost per gel

Problems Advantages

Film & developer costs No film or developer solutions necessary

Antibody dilutions More dilute secondary antibodies can be used

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Molecular weight markers Much more dilute molecular weight markers can be used (I use 1µl)

Further advantages

Ability to Multiplex

Problems Advantages

Inability to multiplex with ECL Fluorescence offers the ability to investigate both phosphorylated or modulated forms of proteins on the same gel

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same gel

Stripping and reprobing No requirement to strip and reprobe –avoiding associated issues

Inaccurate normalization More accurate normalization with multiplex fluorescence

Longer time taken for ECL Process is streamlined resulting in quicker results

Tips for making the switch from ECL to fluorescence (1)

• Optimize Primary Antibody concentrations

• Select the dilution that yields the highest

signal-to-background ratio

• When adapting a chemiluminescent

protocol for fluorescent detection, primary

and secondary antibody concentrations

Antibody

• Different blocking buffers can give

different effects on primary antibodies (I

use 5% non-fat milk in PBS-T)

Blocking

• Be wary of particulates in buffers that

Buffer

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and secondary antibody concentrations

may need to be altered (generally lowered

in my experience)

• Use membrane with low autofluorescence

• Low fluorescent PVDF membranes

available

Membrane

• Be wary of particulates in buffers that

can settle on the membrane and create

fluorescent artifacts

• Including biro markers – use a pencil to

mark membranes)

• Membrane should be done with blunt

forceps – sharp objects can also create

artefacts

Membrane Handling

Tips for making the switch from ECL to fluorescence (2)

• Most fluorescent antibodies will not be

photo bleached by normal laboratory

lighting

• Aliquots of antibodies should be kept in

the dark to prevent possible bleaching

Photo bleaching

• Avoid stripping highly abundant proteins

(e.g. actin or beta tubulin)

• Removal of these proteins from the

nitrocellulose membrane is difficult and

may remove proteins from other areas

of the membrane too

Strip/Reprobe

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of the membrane too

• Use powder-free nitrile gloves when

handling the membrane to minimize

artefacts and fingerprints on the blots

Artefacts

• Bromophenol blue dye fronts can

fluoresce – ensure the dye front has

migrated away from the sample

Dyes

Note: Adapted from http://www.bioradiations.com/focus-on-technology/69-western-blotting/1321-transitioning

Suggestions for multiplexing

Use primary antibodies from different species.

Many from similar species (e.g. Rat & mouse) may give

cross-reactivity even if the antibodies are cross-

adsorbed

Use secondary antibodies that have been highly cross-

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Use secondary antibodies that have been highly cross-

adsorbed

Avoid cross-channel fluorescence by using

fluorochromes with distinct spectra

Independently optimize the conditions for each

antibody before attempting to multiplex

Some antibodies will give multiple bands on a blot,

which may appear where you expect your second

antibody to appear

The future of Westerns?

In gel Western Blot

• No transfer steps required

• Actual gel used

• Ideal for hard to transfer proteins

• No blocking steps

• Sensitive and versatile

On / In- cell Western Blot

• Similar to immunocytochemistry

• Fluorescent secondaries used within wells of 96 / 384 well plate

• Can also be used with RNAi

• High sensitivity

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• Sensitive and versatile • High sensitivity

Source: Paguirigan AL et al. Expanding the Available Assays: Adapting and Validating In-Cell Westerns in Microfluidic Devices for Cell-Based Assays.

Assay Drug Dev Technol 8, 591-601 (2010).

Imaging systems available

Non-IR systems

• ChemiDoc MP

• Image Quant LAS 4000

• Typhoon imager

IR systems

LiCor Odyssey system

• Pros

• low autofluorescence in the IR range results in enhanced sensitivity

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• Typhoon imager

• Fluorchem Q and M systems,

• G:BOX (with bolt on)

• Gel Logic 200

• Fusion FX5

sensitivity

• Uses two lasers to excite at 700 and 800nm

• Cons

• Cannot use secondary antibodies for other applications (e.g. IHC, ICC)

Challenges or pitfalls in fluorescent Western blotting – High background

Cause Solution

BSA used for blocking Blocking solutions containing BSA may cause high membrane background –try alternative blocking solution or adding SDS

Not using optimal blocking reagent

Compare different blocking buffers to find the most effective one

Background on nitrocellulose

Add Tween-20 to diluted antibodies to reduce background

Background on PVDF Use low-fluorescent PVDF membranes

Antibody concentrations too Use optimal dilutions for both primary and secondary antibodies

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Antibody concentrations too high

Use optimal dilutions for both primary and secondary antibodies

Cross-reactivity of antibody with contaminants in blocking

Use different blocking buffer – milk contains IgGs which may cross-react with anti-goat secondary antibodies

Inadequate antibody volume Ensure all of the membrane is covered with liquid

Membrane contamination Use forceps when handling membrane, keep handling to a minimum

Challenges or pitfalls in fluorescent Western blotting – background spots

Cause Solution

Membrane not fully wetted Keep membrane completely wet at all times

Contaminated forceps or dishes

Carefully clean forceps and dishes before and after use

Dirty scanning surface Clean the scanner before use

Particulates in buffers Filter home made buffers prior to use to remove any particles

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Challenges or pitfalls in fluorescent Western blotting – Weak signal

Cause Solution

Not using optimal blocking reagent

Compare different blocking buffers to find the most effective one

Insufficient antibody used Increase the amount of primary antibody used. Try extended incubation time. Increase amount of secondary antibody used.

Too much detergent used –signal washed away

Reduce Tween 20 and / or SDS in antibodies – recommended SDS is 0.01-0.02%

Insufficient antigen on membrane

Increase amount of sample loaded onto the gel

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membrane

Poor transfer of protein Check transfer buffer. Try staining gel to ensure proteins not retained.

Protein lost from membrane Reduce blocking time and / or detergent composition

Proteins not retained during transfer

Small proteins may pass through – try smaller pore size membrane or reduce transfer time. Modulate SDS concentration.

Challenges or pitfalls in fluorescent Western blotting – Nonspecific bands

Cause Solution

Antibody concentration too high

Reduce concentration of antibodies used. Reduce incubation times. Increase Tween-20 concentration

Not using optimal blocking solution

Optimize blocking solution

Cross-reactivity between antibodies in a multiplex experiment

• Double-check the sources and specificities of the primary and secondary antibodies used

• Use only highly cross-adsorbed secondary antibodies. There is always

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• Use only highly cross-adsorbed secondary antibodies. There is always potential for cross-reactivity in two-color experiments

• Use less secondary antibody to minimize this

• Always test the two colors on separate blots first so you know what bands to expect and where

• Avoid using mouse and rat antibodies together, if possible. Because the species are so closely related, anti-mouse will react with rat IgG to some extent, and anti-rat with mouse IgG. Sheep and goat antibodies may exhibit the same behavior

Bleed through of signal from one channel into other channel

Check spectra of secondary antibodies used. Reduce signal by decreasing protein loaded or antibody concentration

Note: Adapted from http://www.licor.com/bio/applications/quantitativeWesternBlots/troubleshooting.jsp

Protocols and Resources

Secondary portfolio: ~2600 products

Enzymes

• HRP (over 300 antibodies)

• AP (nearly 300 antibodies)

Fluorochromes

• Alexa Fluor® 488, Cy2®, FITC,

Available conjugates:

• Pre-adsorbed against up to 3 species (rat, mouse, human)

• Pre-adsorbed against up to 8 species (rat/mouse, human, chicken, goat, rabbit, cow, horse, pig)

Affinity purified/pre-adsorbed

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• Alexa Fluor 488, Cy2 , FITC, DyLight® 488

• Cy3®, DyLight® 550, PE, Texas Red®, TRITC

• DyLight® 594

• Alexa Fluor® 647, APC, Cy5®, DyLight® 650

Other

• Biotin

• Gold

pig)

F(ab’)2/whole IgG to:

• IgA (H+L)/Fc

• IgD(H+L)/Fc

• IgE (H+L)/Fc

• IgG (H+L)/Fc

• IgM (H+L)/Fc

Target format/isotype

Brand new: Alexa Fluor® conjugated secondary antibody range

Alexa Fluor® 488Alexa Fluor® 555

Alexa Fluor® 647Alexa Fluor® 594

ab150157 ab150113ab150151

Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

www.abcam.com/alexa

Why to choose an Abcam AlexaFluor® conjugated secondary?

• Extensively tested in the Abcamlaboratories – to guarantee bright staining and low background

• Large selection of pre-adsorbed antibodies-ensuring low species cross-reactivity

• Dilution range of 1/200 – 1/1000 (10-2

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• Dilution range of 1/200 – 1/1000 (10-2 µg/ml) in IF/ICC – perform at least 250 stainings*

• Antibodies conjugated to Alexa Fluor®

488/555/594/647 – for your multi-color imaging experiments

• Competitively priced

Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

Directly conjugated primary antibodies and proteins

• Tandem dyes (APC/Cy7®, PE/Cy5®)

• PE

• Texas Red® ...... and more

• FITC

• APC

• Cy3®

Extensive range of primary antibodies and detection and purification proteins conjugated to various fluorescent labels:

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Refine search for conjugate

EasyLink Antibody Conjugation Kits

• A choice of 18 fluorescent labels

• Rapid labeling protocol

• Available in convenient sizes

• 3x10µg

•

The easiest way to conjugate your antibodies

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• 1x100µg

• 3x100µg

• 1x1mg

Find out more: http://www.abcam.com/EasyLink

Benefits of EasyLink

Eliminates

• Indirect detection methods

• Column separation steps

• Loss of material

• Non-specific binding of secondary reagents

• Additional incubation steps and sample dilutions

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How the kit works

Optiblot – Fluorescence detection kit

Fluorescent western blot kit (ab133410)

Fluorescent detection of TWO proteins on one blot – no need to strip and reprobe

Includes two fluorescently labeled secondary antibodies(RPE & APC) – visualized with Cy3-GREEN and Cy5-RED channels

No need to rely on Infra Red imaging systems

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Allows detection of loading control and protein of interest in one experiment

Includes washing and blocking buffers optimized for fluorescent Westerns

Brighter signal than Cy dyes

Compatible CCD Imaging systems

ChemiDoc MP (Bio-Rad), LAS 4000 and Typhoon (GE), Fluorchem Q and M systems (Protein Simple), G:BOX (Syngene), Gel Logic (Bruker), Fusion FX5 (Vilber) etc...

AFP and CEA proteins probed using Optiblot Fluorescent Western Blot Kit

Optiblot – ECL substrate kits

ECL substrates that exhibit both chemiluminescence and chemifluorescence(e.g. can be imaged with Typhoon Imager)

Optiblot ECL Max(ab133408)

Optiblot ECL Ultra(ab133409)

Sensitivity Detect attomoles of protein

per band

Detect very low-abundance proteins not

detected by other substrates

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Low background High signal to noise ratio with film detection

Quantitative Broad linear range (above 3 orders of magnitude) – detect low and high

abundance proteins in the same exposure

Long lasting signal

Image blots hours after substrate incubation – no need to rush to the

dark room

Versatile Room Temperature Stable for Up to 1 year & compatible with CCD

imagers and film detection

Ex/Em Excitation with blue 488nm laser – detection using orange filter

compatible with emission of Cy3 dye

Optiblot – Western blot reagents

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Find all the western blot reagents optimized for your experiments in one place:

• Electrophoresis gels – simplified loading and long shelf life

• Buffers and accessories – tailored for optimal experiments

• Optiblot Blue - 15 min, rapid Coomassie gel stain

• Bradford reagent and BCA assay

• Protein ladders, ECL kits, low fluorescence PVDF membranes … and more

Find out more: www.abcam.com/Optiblot

More Resources

• Optiblot landing page:

www.abcam.com/Optiblot

• Optiblot FAQS:

www.abcam.com/OptiblotFAQS

• Western blot webinar:

www.abcam.com/webinars

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Western Blot Reagents and Troubleshooting Tips Booklet

www.abcam.com/webinars

• Western blot detailed guide and video:

http://www.abcam.com/westernblotguide

• Loading control guide:

http://www.abcam.com/loadingcontrolguide

• More protocols and guides:

http://www.abcam.com/protocolguides

Promotion

Exclusive webinar promotion on products for fluorescent western blot

• Save on all qualifying products:

• The entire Optiblot range

• All secondary antibodies, Save

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• All secondary antibodies,

• Fluorophore conjugated detection and purification proteins

• Fluorophore conjugated primary antibodies

• Further information to be sent after the webinar

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Note: Promotions may not be available on future viewings of the webinar

Allergy and Asthma 2013

Date: May 23-24, 2013

Venue: Bruges, Belgium

Conference Topics

• Innate immune cells in asthma

• Epithelial biology and asthma

• Understanding adaptive immunity in

Oral abstract deadline:February 22, 2013

Poster abstract deadline:March 25, 2013

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• Understanding adaptive immunity in

asthma

• Environment and asthma

Confirmed speakers

• David Artis (University of Pennsylvania)

• John Fahy (University of California)

• Darryl Knight (University of British

Columbia)

• Carla Ribeiro (University of North Carolina)

and may more....

Meeting website:www.abcam.com/AA2013

Inflammasomes in Health and Disease

Date: June 24-25, 2013

Venue: Boston, US

Conference Topics

• General mechanisms of activation

• Inflammasomes in infection

Oral abstract deadline:April 26, 2013

Poster abstract deadline:May 17, 2013

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• Inflammasomes in infection

• Inflammasomes and sterile inflammation

• Metabolism-inflammasome link

Keynote speaker:

• Vishva Dixit (Genentech, US)

Meeting chairs:

• Kate Fitzgerald, University of Massachusetts

• Gabriel Nunez, University of Michigan

Meeting website:www.abcam.com/inflammasomes2013

Upcoming webinars

IHC/ICC staining techniques using single and multiple labels

March 06, 2013

15:00 GMT, 10:00 EDT, 07:00 PDT

www.abcam.com/IHCICCwebinar

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labels www.abcam.com/IHCICCwebinar

To interact or not to interact? Immunoprecipitateto answer this question

March 21, 2013

15:00 GMT, 10:00 EDT, 07:00 PDT

www.abcam.com/events

Questions?

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