Presentation is loading. Please wait.

Presentation is loading. Please wait.

Rapid Stain-Free Western Blotting with the V3Western Workflow™

Similar presentations


Presentation on theme: "Rapid Stain-Free Western Blotting with the V3Western Workflow™"— Presentation transcript:

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 (www.tolweb.org).) 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


Download ppt "Rapid Stain-Free Western Blotting with the V3Western Workflow™"

Similar presentations


Ads by Google