western blotting

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

Membrane

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

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’

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

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 Detection Indirect Detection

IP = Immunoprecipitation

Indirect Vs. Direct Detection

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®

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

innova biosciences

rights reserved sdspage

membrane gel

indirect detection indirect

western blot experiments

successful western blot

sdspage rig

transfer buffer

Science

principles of immunodetection 2

buffering agents and buffers · [electrophoresis/blotting,...

northern blotting

immunodetection immuno-essais : kits de détection

multi-array™ immunodetection of biowarfare agents using

protein blotting guide - wolfson centre home...

southern blotting

protein analysis - wolfson centre home...

kelompok 6 kimia b (southern blotting dan northern blotting)

human acrosome biogenesis: immunodetection of …...human...

protein blotting

blotting techniques

this is your presentation title - fac.ksu.edu.sa ·...

thermo scientific pierce western blotting handbook and...

western- superstar immunodetection...

maximize your western blotting...

maximize your western blotting results brochure ·...

immuno blotting

principles of immunodetection