Rapid Stain-Free Western Blotting with the
V3Western Workflow™
Biotechnology ExplorerTM Program
Rapid V3 Stain-Free Western Blotting Workshop Outline
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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
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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
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Rapid Western Blotting + V3 Stain-Free
Rapid Blotting
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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
Visualize
Separation
Verify Validate
Transfer Western
• Stain-Free blot imaging
– Validate Western Blot
• Quantitation of results
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Separate
Proteins
Transfer
Proteins
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
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Western Transfer: Electroblotting Methods
 Tank (wet) blotting
– Assemble transfer sandwich
• Includes gel, membrane, filter paper
– Place sandwich in non-conducting
transfer cassette
– Submerge cassette into tank filled
with buffer that conducts electrical current
provided by power supply to mobilize
proteins from gel (cathode [-] side) to
membrane (anode [+] side)
– Large volume of buffer dissipates heat, but
provides more resistance, so transfer takes longer
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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
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Western Transfer: Comparison of Electroblotting Methods
Tank Blotting
Semi-Dry Blotting
Traditional
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Rapid
Transfer time
30 min – overnight
15 – 60 min
3 – 15 min
Handling convenience
Manual assembly of transfer
components
Manual assembly of transfer
components
Prepackaged, presaturated
components
Transfer parameters
Widest range of power settings
and transfer times
Power and transfer time limited
due to lack of cooling options
Preinstalled, customizable
programs, or userprogrammable settings
Temperature control
Cooling with ice pack or
refrigerated water circulator
None
None
Buffer requirement
1-12 L, system dependent
250 ml per blot
No additional buffer required
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Trans-Blot Turbo Rapid Semi-Dry Blotting
– The Easy-Bake oven of western blotting
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Trans-Blot Turbo Rapid Semi-Dry Blotting
– Advantages
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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
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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
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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
58 Da
UV light
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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
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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
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Imaging Capabilities with Gel Doc EZ
– Color coded trays for different uses
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Rapid V3 Stain-Free Western Blotting Lab
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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
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Comparative Proteomics Kit II: Western Blot Module
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Applied immunology activity
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Use antibodies as detection tools
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Laboratory extension to Comparative
Proteomics Kit I: Protein Profiler Module
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Includes sufficient materials for 8 student
workstations
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Obtain fish samples, extract protein,
visualize proteome after SDS-PAGE,
specifically detect myosin light chain
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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:
Catfish
Salmon
Shark
Sturgeon
Trout
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Workflow
Run one gel for staining and blotting
Load extracted fish muscle
extracts on gel
Visualize transfer to
membrane on Gel Doc EZ Imager
Perform immunodetection for myosin light chain
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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 development
Assembling the Mini-PROTEAN Tetra Modules
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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
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Processing the Gel
Cut off wells and foot of gel
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Activating Stain-Free TGX Gels
ImageLab
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Activating Stain-Free TGX Gels
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Stain-Free Gel Imaging
 First level
– Second level
• Third level
– Forth level
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Preparing for Transfer in the Trans-Blot Turbo
One Mini Gel
Top of gel faces upward
Two Mini Gels
Top of gels face outward
Ion transfer stack that includes the membrane goes on the bottom, then
the gel, then the top ion transfer stack. Roll out bubbles!
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Trans-Blot Turbo Transfer
 Settings:
25 V, 2.5 A, 15 min when running 2 gels per tray
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Stain-Free Blot Imaging
 First level
– Second level
• Third level
– Forth level
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Stain-Free Blot Imaging
 First level
– Second level
• Third level
– Forth level
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Stain-Free Blot Imaging
 First level
– Second level
• Third level
– Forth level
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Example Results with Stain-Free Imaging
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Kaleidoscope Standard
Catfish
Salmon
Shark
Sturgeon
Trout
Actin/Myosin Standard
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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
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Western Blotting: Primary Antibody
•Discard blocking solution
Add Primary
Antibody
•Pour 10 ml of primary antibody onto the membrane
and agitate periodically for 10 minutes
•Primary antibody will bind to the myosin light-chain
(anti- myosin light-chain)
Wash
•Quickly rinse membrane in 25 ml of wash buffer and
discard the wash buffer
•Add 25 ml of wash, agitate periodically for 3 minutes
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Western Blotting: Secondary Antibody
Add
Enzyme-linked
Secondary Antibody
•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
Wash
•Quickly rinse membrane in 25 ml of wash buffer and
discard the wash buffer
•Add 25 ml of wash, agitate periodically for 3 minutes
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Western Blotting: Color Development
Add Enzyme Substrate
•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
Watch for Color
Development
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Example Western Results after TBT Transfer
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Kaleidoscope Standard
Catfish
Salmon
Shark
Sturgeon
Trout
Actin/Myosin Standard
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Western Blot Storage
Store Membrane
•Rinse the developed membrane twice with
distilled water and blot dry
•Air dry for 30min-1hr and store in lab notebook
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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
• Damon_Tighe@bio-rad.com
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Click to add title here
 First level
– Second level
• Third level
– Forth level
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