rapid stain-free western blotting with the v3western workflow™

Rapid Stain-Free Western Blotting with the V3Western Workflow™Biotechnology ExplorerTM Program

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Rapid V3 Stain-Free Western Blotting Workshop Outline

Introduction to Rapid Blotting and V3 Stain-Free Protein Gel Electrophoresis Stain-Free Gel Activation Western Transfer Stain-Free Gel/Blot Imaging Block nitrocellulose membrane Incubation with antibody solutions Color development of the blot


Rapid Western Blotting + V3 Stain-Free

A new approach to western blotting workflows

– Rapid• Faster electrophoresis times

• Faster protein transfer times

• Faster protein visualization

– Higher Throughput• More gels transferred in a single blotting unit

– Real Time Monitoring of Experiment using Stain-Free Technology• Monitor protein separation prior to transfer

• Monitor protein transfer prior to blot probing


Rapid Western Blotting + V3 Stain-Free

Rapid Blotting


What is V3 Stain-Free?

Visualize. Verify. Validate.

– Separate Proteins• Electrophoresis with TGX gels

– Visualize Separation• Stain-Free gel imaging

– Transfer Proteins• Trans-Blot Turbo rapid transfer

– Verify Transfer• Stain-Free blot imaging

– Validate Western Blot• Quantitation of results

SeparateProteins

VisualizeSeparation

TransferProteins

VerifyTransfer

ValidateWestern


Western Transfer: Blotting Methods

Methods to transfer proteins to solid support– Microfiltration

• Used to capture proteins that are in solution, utilizes vacuum for protein immobilization onto membrane

• Rapid due to lack of size separation step, but may be less informative

– Diffusion/Capillary• Used to transfer proteins from gels, involves wicking of buffer through

a weighted transfer stack, is very slow and can be inefficient for larger proteins

– Electroblotting• Used to transfer proteins from gels, is much faster than diffusion,

involves electrical current-mediated mobilization of protein through a buffer-saturated transfer stack

• Several varieties including Tank (wet), Semi-Dry, and Rapid Semi-Dry


Western Transfer: Electroblotting Methods

Tank (wet) blotting

– Assemble transfer sandwich• Includes gel, membrane, filter paper

– Place sandwich in non-conductingtransfer cassette

– Submerge cassette into tank filledwith buffer that conducts electrical currentprovided by power supply to mobilizeproteins from gel (cathode [-] side) to membrane (anode [+] side)

– Large volume of buffer dissipates heat, but provides more resistance, so transfer takes longer


Western Transfer: Electroblotting Methods

Semi-Dry blotting

– Assemble transfer sandwich, which is

pre-saturated in transfer buffer

– Distance between electrodes is very small

(only the width of the transfer sandwich)

– Smaller volume of buffer decreases ability to

dissipate heat, but also lowers resistance,

allowing transfer to occur more rapidly


Western Transfer: Comparison of Electroblotting Methods

Transfer time 30 min – overnight 15 – 60 min 3 – 15 min

Handling convenience Manual assembly of transfer Manual assembly of transfer Prepackaged, presaturatedcomponents components components

Temperature control Cooling with ice pack or None Nonerefrigerated water circulator

Transfer parameters Widest range of power settings Power and transfer time limited Preinstalled, customizableand transfer times due to lack of cooling options programs, or user-

programmable settings

Buffer requirement 1-12 L, system dependent 250 ml per blot No additional buffer required

Tank Blotting Semi-Dry Blotting

Traditional Rapid


Trans-Blot Turbo Rapid Semi-Dry Blotting

– The Easy-Bake oven of western blotting


Trans-Blot Turbo Rapid Semi-Dry Blotting

– Advantages• Rapid semi-dry system

• Preassembled membrane packs

• Bulk consumables are in the works

• Individual transfer trays = flexible start times

• Can transfer up to 4 Mini Gels at a time


TGX Gel Technology

– What is TGX?• TGX = Tris Glycine eXtended PAGE gels

• Modification of traditional Laemmli system

– What’s different from traditional SDS-PAGE gels?• Extended shelf life - gels stable for 12 months

• Faster run times, because TGX gels

can withstand higher voltages

• More cost effective than traditional PAGE gels

• Available in Stain-Free version


Stain-Free TGX Gels

– How does Stain-Free chemistry work?• Gels contain a trihalo compound

• Trihalo = triple halogen = 3 Chlorine, Bromine, Fluorine, or Iodine

• UV light activates covalent reaction between trihalo compound and tryptophan residues in proteins

• Reaction adds 58 Da moiety to tryptophans

MV G

SL

WR UV light

MV G

SL

WR

58 Da


Stain-Free TGX Gels

– What’s the result of this reaction?• 58 Da moieties fluoresce under UV light

• Allows protein visualization without staining

– Will adding 58 Da to every tryptophan affect the

apparent weight or mobility of my protein?• UV-induced linkage occurs after electrophoresis,

so protein mobility is not altered


Imaging with the Gel Doc EZ

– Easy to use, can perform a wide range of documentation and quantification functions

• Automatic lane and band detection

• Easy quantification of results

– Auto control of filters, lens, or lights• System automatically focuses, adjusts filter,

determines optimal exposure

– Fast image acquisition with preset

and customizable controls


Imaging Capabilities with Gel Doc EZ

– Color coded trays for different uses


Rapid V3 Stain-Free Western Blotting Lab

– Run samples on Stain-Free TGX gels– Visualize protein separation using Gel Doc EZ– Transfer proteins to nitrocellulose using Trans-Blot Turbo– Verify protein transfer using Gel Doc EZ– Immunodetection for myosin light chain


Comparative Proteomics Kit II: Western Blot Module

Applied immunology activity

Use antibodies as detection tools

Laboratory extension to Comparative

Proteomics Kit I: Protein Profiler Module

Includes sufficient materials for 8 student workstations

Obtain fish samples, extract protein, visualize proteome after SDS-PAGE, specifically detect myosin light chain


Proteome Diversity is an Indicator of Evolutionary Relatedness

Evolutionary tree showing the relationships of eukaryotes. (Figure adapted from the tree of life web page from the University of Arizona (www.tolweb.org).)

Samples today:CatfishSalmonShark SturgeonTrout


Workflow

Load extracted fish muscle extracts on gel

Run one gel for staining and blotting Activate Stain-Free gel to visualize proteins on Gel Doc EZ imager

Transfer proteins from gel to membrane on Trans-Blot Turbo

Watch for color developmentPerform immunodetection for myosin light chain

Visualize transfer to membrane on Gel Doc EZ Imager


Assembling the Mini-PROTEAN Tetra Modules


Loading and Running the Gels

Samples already heated to 95oC in Laemmli buffer

Load 5 ul Kaleidoscope Standard

Load 3 ul fish samples and Actin/Myosin

Run gel 300 V, 18 min


Processing the Gel

Cut off wells and foot of gel


Activating Stain-Free TGX Gels

ImageLab


Activating Stain-Free TGX Gels


Stain-Free Gel Imaging

First level – Second level

• Third level– Forth level

catfis

h

salm

on

shark

sturgeon

troutacti

n/myo

sin


Preparing for Transfer in the Trans-Blot Turbo

One Mini Gel Two Mini Gels

Ion transfer stack that includes the membrane goes on the bottom, then the gel, then the top ion transfer stack. Roll out bubbles!

Top of gel faces upward Top of gels face outward


Trans-Blot Turbo Transfer

Settings:

25 V, 2.5 A, 15 min when running 2 gels per tray

15

15

15


Stain-Free Blot Imaging







catfis

h

salm

on

shark

sturgeon

trout

actin/m

yosin


Example Results with Stain-Free Imaging

1 blank 2 blank 3 Kaleidoscope Standard 4 Catfish 5 Salmon 6 Shark 7 Sturgeon 8 Trout 9 Actin/Myosin Standard10 blank


Western Blotting: Block

Blocking Buffer Remove membrane from the blotting sandwich and immerse in 25ml of blocking solution for 10 minutes

5% non-fat milk: Prevents the primary antibody from binding randomly to the membrane

Phosphate buffered saline (PBS): Provides the correct environment (pH, Salt) to maintain protein shape

0.025% Tween 20: non-ionic detergent that prevents non-specific binding of antibodies to the membrane


Western Blotting: Primary Antibody

Add Primary Antibody

(anti-myosin light-chain)

Wash

•Discard blocking solution

•Pour 10 ml of primary antibody onto the membrane and agitate periodically for 10 minutes

•Primary antibody will bind to the myosin light-chain

•Quickly rinse membrane in 25 ml of wash buffer and discard the wash buffer

•Add 25 ml of wash, agitate periodically for 3 minutes


Western Blotting: Secondary Antibody

Add Enzyme-linked Secondary Antibody

Wash

•Discard wash solution

•Pour 10 ml of the secondary antibody onto the membrane and agitate periodically for 10 minutes

•Secondary antibody will bind to the primary antibody

•Quickly rinse membrane in 25 ml of wash buffer and discard the wash buffer

•Add 25 ml of wash, agitate periodically for 3 minutes


Western Blotting: Color Development

Add Enzyme Substrate

Watch for Color Development

•Discard wash solution

•Add 10 ml of the enzyme substrate (HRP color detection reagent) onto the membrane

•Incubate until color develops, up to overnight at room temperature

•The colorimetric substrate is cleaved by the enzyme conjugated (attached) to the secondary antibody


Example Western Results after TBT Transfer

1 blank 2 blank 3 Kaleidoscope Standard 4 Catfish 5 Salmon 6 Shark 7 Sturgeon 8 Trout 9 Actin/Myosin Standard10 blank


Western Blot Storage

•Rinse the developed membrane twice with distilled water and blot dry

•Air dry for 30min-1hr and store in lab notebook

Store Membrane


Like what you see? Find out more!

Visit us on the web– www.explorer.bio-rad.com

Rapid Western + V3 Stain-Free Workflow

Application Note coming soon!

Bio-Rad Curriculum and Training Specialist– Damon Tighe

• [email protected]


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rapid stain-free western blotting with the v3western workflow™

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weighted transfer stack

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