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

Dr Brian Carpenter
Spent the majority of his scientific
career performing Western Blot
experiments.

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

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

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’

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

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

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

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

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

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

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

Detection Methods
Chemical vs. Immunofluorescence
Substrate
Fluorescent
Label
Excitation Emission
Light
Emission

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

Indirect Vs. Direct Detection

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
Lightning-Link® HRP Anti-prM
Lightning-Link® HRP Anti-E
Direct DetectionIndirect Detection
IP = Immunoprecipitation

Labeled Antibodies
The major problems with labeled antibodies:
• Their lack of availability
• The difficulty of conjugating antibodies yourself by traditional
methods

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

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®

Lightning-Link®

2°Ab Amplification Hypothesis?

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

Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
facebook.com/InnovaBiosciences
@InnovaBioSci
Innova Biosciences

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

Western blotting - the principles and a comparison of indirect vs direct immunodetection

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