Western blotting - the principles and a comparison of indirect vs direct immunodetection
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Transcript of Western blotting - the principles and a comparison of indirect vs direct immunodetection
© Innova Biosciences ltd. 2013. All rights reserved
Western Blotting
the principles and a comparison of indirect vs. direct immunodetection
© Innova Biosciences ltd. 2013. All rights reserved
Dr Brian Carpenter Spent the majority of his scientific career performing Western Blot experiments.
© Innova Biosciences ltd. 2013. All rights reserved
Introduction
Agenda: • Sample preparation • SDS-PAGE • Western Blot transfer • Blocking • Indirect Detection – primary antibody incubation and indirect detection • Indirect vs. Direct antibody detection – advantages and disadvantages • Direct detection for multiplex fluorescent Western blotting • Problems with sourcing labeled antibodies for direct detection • The solution simple - antibody labeling kits • Questioning the secondary antibody amplification hypothesis
© Innova Biosciences ltd. 2013. All rights reserved
Introduction
Setting the scene: • Significant number of webinars discussing the fundamentals of Western
Blotting • Abcam, Novus Biologicals and Proteintech
• Design an informative webinar providing hint and tips to increase chances of obtaining a successful Western Blot based upon my own experiences.
• Ultimate ambition is to create a check list of points to ensure a thorough
understanding of the technology to facilitate troubleshooting
© Innova Biosciences ltd. 2013. All rights reserved
Western Blotting
‘Is like building a car engine – without looking at the instructions, you do not know if it will start or not until all parts are assembled (in what you presume is the right order) and you turn the key’
© Innova Biosciences ltd. 2013. All rights reserved
Sample Preparation
• Research your target protein thoroughly
Expression pattern Cellular localisation Post-translational modification Predicted vs. actual size Review literature + various websites
• Design an effective sample extraction protocol
Choose the right buffer – cytoplasmic vs. membrane bound proteins
Protease inhibitors Phosphatase inhibitors Cool buffers + prepare fresh Clean tools such as homogenisers thoroughly Prepare samples quickly and efficiently Store appropriately
© Innova Biosciences ltd. 2013. All rights reserved
SDS-PAGE
Ensure SDS-PAGE rig is clean Choose the optimal percentage SDS-PAGE gel High vs. low pre-stained molecular weight markers Boiling of samples Run a reasonable amount of protein do not overload –important to
measure protein concentration Balance your lanes to prevent smiling Make sure the SDS-PAGE rig is connected to the power supply correctly Optimise the running conditions - keeping the system cold, low voltage,
overnight vs. 1 hour
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Western Blot Transfer
Ensure transfer apparatus is clean Wear gloves all the time! Do not contaminate the apparatus Wet vs. dry transfer – option depends upon protein Membrane – Immobilon® P my preferred choice. Once activated with
methanol, do not allow the membrane to dry out. Think about using two membranes for smaller proteins Equilibrate membrane in transfer buffer for 5 minutes Filter paper, membrane + gel – assemble in correct order
Gel
Membrane
-ve
+ve
Filter paper
Immobilon® is a registered trademark – Merck kGaA, Darmstadt, Germany
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Western Blot Transfer
Roll out any trapped bubbles within the system Assemble the transfer apparatus following the manufacturer’s protocol Connect transfer system to power pack – remember proteins always
migrate to the positive - overnight wet transfer - 40V Confirm the transfer worked – stain membrane with ponceau S + gel with
coomassie blue
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Blocking
Never allow the membrane to dry out – keep hydrated Buffer of choice – PBS vs. TBS Use non-fat dry milk or BSA
Make sure it is all dissolved Store blocking solution in fridge – do not leave at
room temperature overnight Recommended blocking time 1 hour at room temperature Agitate gently using a rocking platform Remember to use gloves and tweezers
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Primary Ab
• Primary Antibody
Optimise antibody dilution – starting point 1 in 1000 Buffer optimisation
• PBS vs. TBS • Milk vs. BSA • + or – Tween • Preferred starting point (TBST + 5% milk)
Incubation period • 1 hour at room temperature • Overnight at 4°C
• Washing
Large volume of blocking buffer – 1cm2 = 1ml Agitation at room temperature 6 x 10minutes
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Secondary Ab
• Secondary Antibody
Starting dilution 1 in 10,000 Secondary antibody
• HRP conjugated – do not add azide to your buffer • Fluorescent label – incubate in the dark
• Washing
Large volume of blocking buffer – 1cm2 = 1ml Agitation at room temperature 6 x 10minutes
• Detection
Remove excess wash buffer using paper towel HRP labeled - ECL + film Fluorescently labeled – use appropriate machine; keep in dark to avoid
quenching
A framework for Western blot trouble shooting
© Innova Biosciences ltd. 2013. All rights reserved
Western Blotting
‘Is like building a car engine – without looking at the instructions, you do not know if it will start or not until all parts are assembled (in what you presume is the right order) and you turn the key’
© Innova Biosciences ltd. 2013. All rights reserved
Detection Methods
Chemical vs. Immunofluorescence
Substrate
Fluorescent Label
Excitation Emission
Light Emission
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Bates DO, Mavrou A, Qiu Y, Carter JG, et al. (2013) Detection of
VEGF-Axxxb Isoforms in Human Tissues. PLoS ONE 8(7):
e68399. doi:10.1371/journal.pone.0068399
http://www.plosone.org/article/info:doi/10.1371/journal.pone.006
8399
Fluorescent Western Blotting
B-actin
Eaton SL, Roche SL, Llavero Hurtado M, Oldknow KJ, et al. (2013)
Total Protein Analysis as a Reliable Loading Control for Quantitative
Fluorescent Western Blotting. PLoS ONE 8(8): e72457.
doi:10.1371/journal.pone.0072457
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072457
© Innova Biosciences ltd. 2013. All rights reserved
Indirect Vs. Direct Detection
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IP – Indirect vs. Direct Detection
Chan AHY, Tan HC, Chow AY, Lim APC, et al. (2012) A Human PrM
Antibody That Recognizes a Novel Cryptic Epitope on Dengue E
Glycoprotein. PLoS ONE 7(4): e33451. doi:10.1371/journal.pone.0033451
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033451
Lightning-Link® HRP Anti-prM
Lightning-Link® HRP Anti-E
Direct Detection Indirect Detection
IP = Immunoprecipitation
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Indirect Vs. Direct Detection
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Labeled Antibodies
The major problems with labeled antibodies: • Their lack of availability • The difficulty of conjugating antibodies yourself by traditional
methods
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What is Lightning-Link® technology?
The worlds fastest, easiest to use and most efficient conjugation technology!
• Only 30 seconds hands-on time! • Over 50 labels available including: Enzymes, fluorescent proteins / dyes, tandems, biotin & streptavidin
Antibodies – Proteins – Peptides
Fast – Easy-to-use – Reliable
Simple Antibody Labeling Kits
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What is Lightning-Link® technology?
• Chemistry expertise not required • 100% antibody recovery
• Pack sizes range from 10ug, 100ug up to 5+mg
• Covalent conjugation ensures long-term stability
• Two ranges Lightning-Link® (3 hours incubation) and Lightning-Link® RAPID
(15 minutes)
Lightning-Link®
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Lightning-Link®
© Innova Biosciences ltd. 2013. All rights reserved
2°Ab Amplification Hypothesis?
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Summary
• Discussed a troubleshooting framework for Western blotting • Indirect vs. direct detection
• Benefits of direct detection
• Problems of sourcing labeled antibodies or labeling antibodies using standard methodology
• The solution - Lightning-Link® simple antibody labeling kits
• 2° antibody amplification hypothesis
© Innova Biosciences ltd. 2013. All rights reserved
Contact
If you would like any more information, please contact us at [email protected]
facebook.com/InnovaBiosciences
@InnovaBioSci
Innova Biosciences
© Innova Biosciences ltd. 2013. All rights reserved
Innova Biosciences Ltd.
Babraham Research Campus,
Cambridge, UK,
CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a registered trademark of Innova Biosciences DyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries