Rapid Stain-Free Western Blotting with the V3Western Workflow™

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

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 Blotting Rapid Western Blotting + V3 Stain-Free

5. Visualize Separation Verify Transfer Validate Western Separate Proteins Transfer Proteins 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 WesternBlot • Quantitation of results

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

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

9. Tank Blotting Semi-Dry Blotting Traditional Rapid Transfer time 30 min – overnight 15 – 60 min 3 – 15 min Handling convenience Manual assembly of transfer Manual assembly of transfer Prepackaged, presaturated components components components 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 Temperature control Cooling with ice pack or None None refrigerated water circulator Buffer requirement 1-12 L, system dependent 250 ml per blot No additional buffer required Western Transfer: Comparison of Electroblotting Methods

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. 58 Da R R W W L L UV light S S M M G G V V 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

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 (www.tolweb.org).) Samples today: Catfish Salmon Shark Sturgeon Trout

20. Activate Stain-Free gel to visualize proteins on Gel Doc EZ imager Run one gel for staining and blotting Load extracted fish muscle extracts on gel Perform immunodetection for myosin light chain Watch for color development Transfer proteins from gel to membrane on Trans-Blot Turbo Workflow Visualize transfer to membrane on Gel Doc EZ Imager

21. Assembling the Mini-PROTEAN Tetra Modules

22. Loading and Running the Gels • Samples alreadyheated 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 sturgeon salmon catfish shark 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. 15 15 15 Trans-Blot Turbo Transfer • Settings: 25 V, 2.5 A, 15 min when running 2 gels per tray

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 sturgeon salmon catfish 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 AntibodyWash

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 • 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

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