1. Rapid Stain-Free Western Blotting with the V3Western Workflow™
Biotechnology ExplorerTM Program
Express Westerns – Rapid

2. 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

3. 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

4. Rapid Western Blotting + V3 Stain-Free
Rapid Blotting

5. 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
Separate
Proteins
Visualize
Separation
Transfer
Verify
Validate
Western

6. 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

7. 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
More cooling capacity, more resistance, slower

8. 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
Heat up more quickly, less resistance, faster

9. Western Transfer: Comparison of Electroblotting Methods
Transfer time 30 min – overnight – 60 min – 15 min
Handling convenience Manual assembly of transfer Manual assembly of transfer Prepackaged, presaturated
components components components
Temperature control Cooling with ice pack or None None
refrigerated water circulator
Transfer parameters Widest range of power settings Power and transfer time limited Preinstalled, customizable
and transfer times due to lack of cooling options programs, or user-
programmable settings
Buffer requirement 1-12 L, system dependent ml per blot No additional buffer required
Tank Blotting Semi-Dry Blotting
Traditional Rapid

10. Trans-Blot Turbo Rapid Semi-Dry Blotting
The Easy-Bake oven of western blotting

11. 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

12. 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

13. Stain-Free TGX Gels How does Stain-Free chemistry work? UV light
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 M V G S L W R
UV light
58 Da

14. 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

15. 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

16. Imaging Capabilities with Gel Doc EZ
Color coded trays for different uses

17. 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

18. 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

19. 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 (
Samples today:
Catfish
Salmon
Shark
Sturgeon
Trout

20. Workflow Run one gel for staining and blotting
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
Visualize transfer to
membrane on Gel Doc EZ Imager
Perform immunodetection for myosin light chain
Watch for color development

21. Assembling the Mini-PROTEAN Tetra Modules

22. 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

23. Processing the Gel
Cut off wells and foot of gel

24. Activating Stain-Free TGX Gels
ImageLab

25. Activating Stain-Free TGX Gels

26. Stain-Free Gel Imaging
First level
Second level
Third level
Forth level
actin/myosin
catfish
salmon
shark
sturgeon
trout

27. Preparing for Transfer in the Trans-Blot Turbo
One Mini Gel Two Mini Gels
Top of gel faces upward 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!

28. Trans-Blot Turbo Transfer
Settings:
25 V, 2.5 A, 15 min when running 2 gels per tray 15

29. Stain-Free Blot Imaging
First level
Second level
Third level
Forth level

30. Stain-Free Blot Imaging
First level
Second level
Third level
Forth level

31. Stain-Free Blot Imaging
First level
Second level
Third level
Forth level
actin/myosin
catfish
salmon
sturgeon
shark
trout

32. 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 Standard
10 blank

33. 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

34. Western Blotting: Primary Antibody
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
Add Primary Antibody (anti- myosin light-chain) Wash

35. Western Blotting: 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
Quickly rinse membrane in 25 ml of wash buffer and discard the wash buffer
Add 25 ml of wash, agitate periodically for 3 minutes
Add Enzyme-linked Secondary Antibody Wash

36. Western Blotting: 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
Add Enzyme Substrate Watch for Color Development

37. 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 Standard
10 blank

38. 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

39. Like what you see? Find out more!
Visit us on the web
Rapid Western + V3 Stain-Free Workflow
Application Note coming soon!
Bio-Rad Curriculum and Training Specialist
Damon Tighe

40. Click to add title here First level Second level Third level
Forth level