Table of Contents
Subscribe to Our Youtube Channel
Related Manuals for ThermoFisher Scientific Invitrogen iBlot 3
Summary of Contents for ThermoFisher Scientific Invitrogen iBlot 3
- Page 1 iBlot 3 Western Blot Transfer System ™ USER GUIDE For dry electroblotting of proteins from mini and midi gels Catalog Number IB31001 Publication Number MAN0028464 Revision A.0 For Research Use Only. Not for use in diagnostic procedures.
- Page 2 Life Technologies Holdings Pte Ltd | Block 33 | Marsiling Industrial Estate Road 3 | #07-06, Singapore 739256 For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition. Revision history: MAN0028464 A.0 (English) Revision Date Description 5 January 2023 New document for iBlot 3 Western Blot Transfer System.
-
Page 3: Table Of Contents
Contents ■ CHAPTER 1 Product information ..........6 Product contents . - Page 4 Contents Operating the iBlot 3 Western Blot Transfer Device ....... . 18 ™...
- Page 5 Contents ■ APPENDIX D Maintenance ............37 Cleaning .
-
Page 6: Chapter 1 Product Information
Product information Product contents iBlot 3 Western Blot Transfer Device contents ™ This manual is intended to provide instructions for the operation of the iBlot 3 Western Blot Transfer ™ Device (Cat. No.IB31001). The contents of the iBlot 3 Western Blot Transfer Device are listed below. See “iBlot 3 Western Blot ™... -
Page 7: Installing The Instrument
Chapter 1 Product information Product contents The iBlot 3 Western Blot Transfer Device complies with the TUV Rhineland North America Inc. safety ™ requirements, part 15 of the FCC rules, and the European Community Safety requirements. Operation of the iBlot 3 Western Blot Transfer Device is subject to the conditions described in this manual. ™... -
Page 8: Service Operation Requirements
Chapter 1 Product information Unpacking instructions Service operation requirements In the event of an equipment malfunction, it is the responsibility of the customer to report the need for service to Thermo Fisher Scientific or to one of the authorized agents. For service information, contact Technical Support. -
Page 9: About The System
Chapter 1 Product information About the system About the system iBlot 3 Western Blot Transfer System ™ The iBlot 3 Western Blot Transfer System consists of the iBlot 3 Western Blot Transfer Device and ™ ™ associated iBlot 3 Transfer Stacks (sold separately). The iBlot 3 Western Blot Transfer Device has a ™... - Page 10 Chapter 1 Product information About the system Schematic of iBlot 3 Transfer Stack showing the flow of current ™ Top felt with folded aluminum tab Copper cathode Cathode gel matrix Plastic divider Membrane (NC or PVDF) Improved anode gel matrix Copper anode Bottom felt Transfer tray After the stack is assembled on the iBlot...
-
Page 11: Description Of Parts
Chapter 1 Product information Description of parts Description of parts iBlot 3 Western Blot Transfer Device ™ The iBlot 3 Western Blot Transfer Device is a protein transfer device with an integrated power supply ™ capable of producing currents up to 6.3 amp, and supplying voltage up to 35 V. Four printed circuit boards hold the electronic components required to process the systems logic unit, modify voltage and currents for display, and power the blotting process. -
Page 12: Lids And Cooling
Chapter 1 Product information Description of parts Lids and cooling The lids of the iBlot 3 Western Blot Transfer Device exert even pressure on the stack surface when the ™ lid is closed. The lids contain a cooling system in the form of a heat sink connected to, and in thermal communication, with the cathode plate. -
Page 13: Guidelines For Iblot ™ 3 Transfer Stacks
Chapter 1 Product information Description of parts Guidelines for iBlot 3 Transfer Stacks ™ • Store the iBlot 3 Transfer Stacks at room temperature. For best results, use the transfer stack ™ before the expiration date printed on the package for each stack. •... -
Page 14: Top Stack
Chapter 1 Product information Description of parts Top stack The Top Stack is separated from the Bottom Stack by a white plastic divider and contains a copper electrode and a transfer cathode gel layer. The transfer gel layer acts as an ion reservoir and is composed of an optimized, proprietary gel composition. -
Page 15: Bottom Stack
Chapter 1 Product information Description of parts Bottom stack The Bottom Stack in the plastic tray contains an absorbent pad, a copper electrode, transfer anode gel layer, and a nitrocellulose or PVDF membrane for protein transfer. The transfer gel layer acts as an ion reservoir and is composed of an optimized, proprietary gel composition. -
Page 16: Transfer Membrane
Chapter 1 Product information Description of parts Transfer membrane The iBlot 3 Transfer Stacks are assembled with the transfer membrane and are available with: ™ • Nitrocellulose membrane (0.2 µm) The nitrocellulose membrane is composed of 100% pure nitrocellulose to provide high-quality transfer. The membrane is compatible with commonly used detection methods such as staining, immunodetection, fluorescence, or radiolabeling. -
Page 17: Iblot ™ Filter Paper
Chapter 1 Product information Description of parts iBlot Filter Paper ™ The iBlot Filter Paper is supplied in two sizes for efficient blotting of mini or midi gels. Briefly wetted in ™ deionized water, the iBlot Filter Paper is placed on top of the pre-run gel before placing the Top Stack ™... -
Page 18: Operating The Iblot
Chapter 1 Product information Operating the iBlot 3 Western Blot Transfer Device ™ Operating the iBlot 3 Western Blot Transfer Device ™ First time usage of the iBlot 3 Western Blot Transfer Device ™ The first time the iBlot 3 Western Blot Transfer Device is turned on, you will need to perform the ™... -
Page 19: Set The Date And Time
Chapter 1 Product information Operating the iBlot 3 Western Blot Transfer Device ™ Set the date and time 1. Press Accept on the End User License Agreement screen to access the Date & Time screen. 2. Touch the Date & Time screen to modify the Time Zone. 3. -
Page 20: Description Of Methods
Chapter 1 Product information Description of methods Description of methods Methods The iBlot 3 Western Blot Transfer Device is pre-programmed with 3 transfer methods using different ™ combinations of voltage, time, and cooling. Transfer Method Run Parameters Broad Range (30–250 kDa) 25 V, 6 mins, Low Cooling High Molecular Range (150–400 kDa) 30 V, 8 mins, No Cooling Low Molecular Range (10–120 kDa) - Page 21 Chapter 1 Product information Description of methods Table 3 Additional recommended parameters for protein transfer with a molecular weight >150 kDa Transfer Stack Volts Transfer Time Cooling Midi transfer stacks 25–30 V 8–10 mins No cooling (2 mini gels or 1 midi gel) Mini transfer stack 25–30 V 6–8 mins No cooling...
-
Page 22: Chapter 2 Protein Transfer Protocol
Protein transfer protocol Experimental overview Experimental outline The table below outlines the experimental steps necessary to perform western blotting using the iBlot ™ 3 Western Blot Transfer Device. For more details on each step, see indicated pages. Step Action Page Select Method for performing transfer. -
Page 23: Recommended Gel Types
Chapter 2 Protein transfer protocol Using the iBlot 3 Western Blot Transfer System ™ • Do not allow the stack to stay in the device for more than 5 minutes after the transfer. Excess time in the device can cause membrane discoloration and higher background. •... -
Page 24: Select A Method
Chapter 2 Protein transfer protocol Using the iBlot 3 Western Blot Transfer System ™ Select a method Select the appropriate Method for your application on the iBlot 3 Western Blot Transfer Device prior to ™ assembling an iBlot 3 Transfer Stack with your gel. ™... - Page 25 Chapter 2 Protein transfer protocol Using the iBlot 3 Western Blot Transfer System ™ 5. After completion of electrophoresis, remove the gel from the cassette. Cut off the gel foot (if present) and well fingers and immerse the gel briefly in deionized water (5–15 seconds) to facilitate easy positioning of the gel on top of the transfer membrane.
-
Page 26: Perform Blotting
Chapter 2 Protein transfer protocol Using the iBlot 3 Western Blot Transfer System ™ 9. Place the tray with the assembled stack layers directly on the blotting surface. Align the copper tab of the tray with the symbol (two possible locations per each station). Do not push the tray too hard as this may elevate and twist the tray or interfere with the contacts. - Page 27 Chapter 2 Protein transfer protocol Using the iBlot 3 Western Blot Transfer System ™ 4. Remove the transfer membrane from the stack and proceed with downstream processing or post-transfer analysis (see “Post transfer analysis” on page 35 for details). Note: PVDF membrane dries quickly. Upon removal from the stack, immediately place the membrane into deionized water.
-
Page 28: Chapter 3 Custom Methods
Custom methods Create custom methods Create a new method Custom methods can be created and saved on the iBlot 3 Western Blot Transfer Device for specific ™ applications or fine-tuning transfer conditions. Custom Methods can be created by editing a pre- programmed method or by creating a new method. - Page 29 Chapter 3 Custom methods Create custom methods 6. Save the newly created custom method by entering the Method name and method description in the Comments field, and touch Save. 7. Touch Close. Note: If the name with the same file name exists, a warning message will appear to save the method as a new file or overwrite the existing file.
-
Page 30: Edit Existing Methods
Chapter 3 Custom methods Create custom methods 8. Touch Start run to begin the transfer. The new saved method will be listed in All and Custom Methods. Edit existing methods Custom methods can be created and saved on the iBlot 3 Western Blot Transfer Device for specific ™... -
Page 31: Create Custom Methods From An Existing Template (Up To 10 Steps)
Chapter 3 Custom methods Create custom methods 9. At the end of the transfer, the current automatically shuts off and the device signals the end of the transfer with repeated alert sounds and a message on the display. Touch Done to stop the alert sounds. -
Page 32: Appendix A Troubleshooting
Troubleshooting Introduction Review the information below to troubleshoot your experiments using the iBlot 3 Western Blot Transfer ™ Device and iBlot 3 Transfer Stack. ™ To troubleshoot the immunodetection process, see the instructions supplied by the manufacturer of the immunodetection reagents. Observation Possible cause Recommended action... - Page 33 Appendix A Troubleshooting Introduction Observation Possible cause Recommended action Difficulty closing lid The transfer stack was too The iBlot 3 Western Blot Transfer Device ™ thick. can only support gels of ≤1.5mm thickness. Note: Do not press forcibly on Ensure the proper gel is being used. the lid as it can cause damage Ensure that additional filter pads have not to the unit.
- Page 34 Appendix A Troubleshooting Introduction Observation Possible cause Recommended action Protein bands distorted on Non-uniform electric field Ensure the gel is properly flattened using the membrane created around wells. Blotting Roller. See “General guidelines” page 22 to obtain good results. Protein not binding/transferring The PVDF membrane was Regions where PVDF membranes are dry to membrane (PVDF) dry/partially dry.
-
Page 35: Appendix B Post Transfer Analysis
Post transfer analysis Post transfer analysis After the transfer, proceed to immunodetection, store the membrane for future use, or stain the membrane. • For immunodetection of proteins, use any Thermo Fisher Scientific immunodetection kit. Note: When using the iBlot 3 Western Blot Transfer System to transfer proteins from SDS-PAGE ™... -
Page 36: Appendix C Optimizing Blotting
Optimizing blotting Optimizing blotting When using the iBlot 3 Western Blot Transfer Device, most proteins transfer efficiently using the ™ protocol in this manual. Based on specific properties of a protein or a set of proteins, some optimization of the blotting protocol may be necessary. Optimize blotting as follows: Perform a water equilibration step before transfer To improve the transfer of high-molecular weight proteins from mini- or midi-NuPAGE... -
Page 37: Appendix D Maintenance
Maintenance Cleaning • Before cleaning the iBlot 3 Western Blot Transfer Device, make sure the device is turned off. ™ • Wipe off the blotting surface and electrodes with a damp cloth or paper tissue. General maintenance • To avoid damaging the iBlot 2 Gel Transfer Device, do not perform any repairs or service other ™... - Page 38 Appendix D Maintenance Replacing the fuse CAUTION! For continued protection against the risk of fire, replace fuses only with listed and certified fuses of the same type and rating as those currently in the device. 1. Power OFF and then unplug the device. 2.
-
Page 39: Replacing Electrical Contacts
Appendix D Maintenance Replacing electrical contacts 6. Replace each failed fuse with a 12.5 A, Time-Lag T, 250 VAC, 5 × 20-mm fuse. Note: The voltage and amperage ratings are on the fuse holder. Ensure the correct orientation of the fuse in the holder (as shown in image below). 7. - Page 40 Appendix D Maintenance Replacing electrical contacts 2. Hold the lid with one hand, then pull out the white rubber seal. 3. Using the screwdriver, unscrew the 2 screws holding the contact. Discard the old screw and contact. 4. Clean the area behind the contact with a damp tissue or cloth. 5.
-
Page 41: Appendix E Product Specifications
Product specifications iBlot 3 Western Blot Transfer Device specifications ™ Dimensions: 44 cm (l) × 30 cm (w) × 18.25 cm (h) Weight: 8.3 kg Electrical Parameters: ~100–240 VAC, 50/60 Hz, 530VA Built-in Features: Digital display, alarm Compatibility: Suitable for transfer of mini- (8 × 8 cm), midi- (8 × 13 cm) iBlot 3 Materials: PC/ABS, Nylon PA12+GF30, Beryllium copper with gold plating, Stainless Steel,... -
Page 42: Iblot ™ 3 Transfer Stack Specifications
Appendix E Product specifications iBlot 3 Transfer Stack specifications ™ iBlot 3 Transfer Stack specifications ™ Specifications for the iBlot 3 Transfer Stacks are listed below. For a more detailed description of the ™ iBlot 3 Transfer Stacks, see “iBlot 3 Transfer Stacks” page 43. -
Page 43: Appendix F Accessory Products
Accessory products iBlot 3 Transfer Stacks ™ iBlot 3 Transfer Stacks are available at thermofisher.com. Ordering information is provided below. ™ Product Quantity Cat. No. iBlot 3 Transfer Stack, Midi, Nitrocellulose 1 pack of 10 IB33001 ™ iBlot 3 Transfer Stack, Midi, PVDF 1 pack of 10 IB34001 ™... -
Page 44: Precast Gels And Premade Buffers
Appendix F Accessory products Precast gels and premade buffers (continued) Product Quantity Cat. No. MagicMark XP Western Protein Standard 250 µL LC5602 ™ SYPRO Ruby Protein Blot Stain 200 mL S-11791 ™ ™ Pierce Reversible Protein Stain Kit for Nitrocellulose Membranes 1.5 L 24580 ™... -
Page 45: Appendix G Safety
Safety WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document. -
Page 46: Control And Connection Symbols
Appendix G Safety Symbols on this instrument Control and connection symbols Symbols and descriptions On (Power) Off (Power) Earth (ground) terminal Protective conductor terminal (main ground) Direct current Alternating current Both direct and alternating current iBlot 3 Western Blot Transfer System User Guide ™... -
Page 47: Safety Information For Instruments Not Manufactured By Thermo Fisher Scientific
Appendix G Safety Safety information for instruments not manufactured by Thermo Fisher Scientific Safety information for instruments not manufactured by Thermo Fisher Scientific Some of the accessories provided as part of the instrument system are not designed or built by Thermo Fisher Scientific. Consult the manufacturer's documentation for the information needed for the safe use of these products. -
Page 48: Cleaning And Decontamination
Appendix G Safety Safety and electromagnetic compatibility (EMC) standards Cleaning and decontamination CAUTION! Cleaning and Decontamination. Use only the cleaning and decontamination methods that are specified in the manufacturer user documentation. It is the responsibility of the operator (or other responsible person) to ensure that the following requirements are met: ·... -
Page 49: Safety Standards
Appendix G Safety Safety and electromagnetic compatibility (EMC) standards (continued) Symbol Information The WEEE (Waste Electrical and Electronic Equipment) symbol indicates that this product should not be disposed of in unsorted municipal waste. Follow local municipal waste ordinances for proper disposal provisions to reduce the environmental impact of WEEE. Visit http://www.lifetechnologies.com/weee for collection and recycling options. -
Page 50: Environmental Design Standards
Appendix G Safety Safety and electromagnetic compatibility (EMC) standards Environmental design standards Reference Description Directive 2012/19/EU European Union “WEEE Directive” — Waste electrical and electronic equipment Directive 2011/65/EU European Union “RoHS Directive” — Restriction of hazardous substances in electrical and electronic equipment Regulation EC European Union “REACH”... -
Page 51: Chemical Safety
Appendix G Safety Chemical safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions: · Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. -
Page 52: Appendix H Documentation And Support
Documentation and support Customer and technical support Visit thermofisher.com/support for the latest service and support information. • Worldwide contact telephone numbers • Product support information – Product FAQs – Software, patches, and updates – Training for many applications and instruments •... - Page 54 iBlot 3 Western Blot System_UG_MAN0028464-v4-GUID-1768287A-604E-443F-8632- BBE6C1271A5C-2023/01/05 19:51:30 en 19:54:10.196Z thermofisher.com/support | thermofisher.com/askaquestion thermofisher.com 5 January 2023...
Need help?
Do you have a question about the Invitrogen iBlot 3 and is the answer not in the manual?
Questions and answers