Introduction

The iBlot® 7-Minute Blotting System consists of the iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stacks that allows you to quickly and reliably perform western blotting of proteins from various types of gels without the need to prepare buffers.

The unique design of the iBlot™ Gel Transfer Device with an integrated power supply, combined with the patented gel matrix technology of the iBlot™ Gel Transfer Stacks generates high field strengths to allow for fast, dry blotting of proteins within 7 minutes. There is no need for an external power supply or to prepare buffers, resulting in consistent performance.

The proteins transferred using the iBlot® 7-Minute Blotting System exhibit higher immunodetection sensitivity when compared to proteins transferred using conventional semi-dry or semi-wet blotting methods.

See below to understand how the iBlot® 7-Minute Blotting System works and Description of Parts section for details on various parts of the system.
 
System Components

The iBlot® 7-Minute Blotting System consists of:

  • iBlot™ Gel Transfer Device: The iBlot™ Gel Transfer Device is a self-contained blotting unit with an integrated power supply that allows for fast, dry blotting of proteins.
  • iBlot™ Gel Transfer Stacks: The iBlot™ Gel Transfer Stacks are disposable stacks with an integrated nitrocellulose transfer membrane to perform dry blotting of proteins. Each iBlot™ Gel Transfer Stack contains a copper electrode and appropriate cathode and anode buffers in the gel matrix to allow fast, reliable dry blotting of proteins without the need to prepare buffers. 

 
Features

Important features of the iBlot® 7-Minute Blotting System are listed below:

  • User-friendly iBlot™ Gel Transfer Device design with an integrated power supply for fast, reliable protein transfer within 7 minutes
  • Ability to perform blotting of E-PAGE™, mini, and midi gels
  • Unique, iBlot™ Gel Transfer Stacks with integrated nitrocellulose transfer membrane allow dry electroblotting of proteins without the need to prepare buffers, and are compatible for use with NuPAGE® Bis-Tris and Tris-Acetate, Tris-Glycine, Tricine, and E-PAGE™ gels
  • Pre-programmed (iBlot™ Gel Transfer Device) with 5 programs for transfer of proteins from various gel types
  • Dry blotting enables higher immunodetection sensitivity
  • Built-in safety features in the device enhance user safety

 
System Overview

The iBlot® 7-Minute Blotting System is based on the dry blotting concept which utilizes the unique, patented gel matrix technology developed by Invitrogen for E-Gel® and E-PAGE™ gels.  To use the iBlot® 7-Minute Blotting System for protein transfer, you will assemble the iBlot™ Gel Transfer Stacks containing the nitrocellulose transfer membrane with your pre-electrophoresed gel on the iBlot™ Gel Transfer Device. Any trapped air bubbles that interfere with efficient protein transfer are removed using the De-bubbling Roller (for E-PAGE™ gels) or using the Blotting Roller (for mini or midi gels). The blotting is performed using a program specific for the gel types.

The following features of the iBlot® 7-Minute Blotting System allow rapid protein transfer without the need for external power supply or pre-made buffers:

  • The iBlot™ Gel Transfer Stacks act as ion reservoirs that contain the appropriate anode and cathode buffers incorporated into the gel matrix, eliminating the need for pre-made buffers or soaked filter papers, and minimizing handling resulting in consistent performance. The iBlot™ Gel Transfer Stacks also contain the copper electrodes (anode and cathode) required for electrophoresis. The protein transfer consistency is increased since the copper anode does not generate oxygen gas as a result of water electrolysis, as compared to conventional inert electrodes present in other blotting systems. See figure below.
  • The design of the iBlot™ Gel Transfer Device reduces the distance between the electrodes and the integrated power supply. This unique design combined with the gel matrix technology of the iBlot™ Gel Transfer Stacks allows the system to generate high field strength and high protein currents increasing the transfer speed.


Schematic of iBlot® 7-Minute Blotting System showing the flow of current




Purpose of the Protocol

This protocol provides the following information:

  • Overview of the dry blotting process to transfer proteins
  • Details and specifications on the iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stacks
  • Protocol to perform blotting using the iBlot™ Gel Transfer Device with the De-bubbling Roller or Blotting Roller
  • Disassembling the iBlot™ Gel Transfer Device
  • Tips on optimizing blotting
  • Examples of expected results
  • Troubleshooting


Immunodetection protocols are not included in this protocol.


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Ordering Information

Sku Name Size Price Qty
LC2100 Blotting Roller, 8.6 cm wide 1 unit USD 30.60
IB301001 iBlot® Transfer Stack, nitrocellulose, regular size 10 sets USD 184.00
IB301002 iBlot® Transfer Stack, nitrocellulose, mini 10 sets USD 149.00

iBlot™ Gel Transfer Device

Front View of iBlot™ Device

The front top view showing various parts of the iBlot™ Gel Transfer Device is shown below.




Rear View of iBlot™ Device


The rear view showing various parts of the iBlot™ Gel Transfer Device is shown below




Control Panel of iBlot™ Device

The control panel of the iBlot™ Gel Transfer Device is described below.

The Digital Display shows six digits that specify the transfer conditions as follows:

  • First two digits indicate the program name
  • Remaining four digits specify the time of transfer in minutes and seconds, respectively.


The Select Button is used to toggle between program and time.
The Up/Down (+/-) Buttons are used to increase or decrease the program, or time. See details on selecting the program.



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Description of Parts

Introduction

The various parts of the iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stacks are described below.
 
iBlot™ Gel Transfer Device


The iBlot™ Gel Transfer Device is a blotting device with an integrated power supply capable of producing currents up to 5.5 amp at 25 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. A pre-installed firmware controls the parameters such as voltage and time, and allows selection of programs (see below for details on each program).
 
 
A top view of an open iBlot™ Gel Transfer Device identifying various parts is shown below.



 



The blotting surface is the area where the iBlot™ Gel Transfer Stacks are placed with the gel to perform blotting. This area also contains the Gel Barriers that guide the proper placement of the transfer stacks to allow correct electrical contact.

De-Bubbling Surface

The de-bubbling surface is the area where de-bubbling of E-PAGE™ gels is performed using the De-bubbling Roller. This area contains Metal Spacers 1 and 2, and hinges to attach the De-bubbling Roller. Barriers are also present on the de-bubbling surface to guide the proper placement of the iBlot™ Anode Stack, Bottom and the gel to allow efficient de-bubbling. The iBlot™ Anode Stack, Bottom is assembled with the gel, Metal Spacers 1 and 2, the iBlot™ Cathode Stack, Top, and the De-bubbling Roller. The entire assembled transfer stack with the gel is pulled together with the pull tab towards the blotting surface resulting in removal of any trapped air bubbles between the gel and the blotting membrane.
 
 Lid

The iBlot™ Lid contains ventilation holes to allow for proper ventilation of the unit during the run. The iBlot™ Disposable Sponge is placed on the inner side of the iBlot™ Lid within the small protrusions present on the lid that allow proper placement of the sponge. The Lid also contains the electrical contacts for the copper electrodes on the stack to complete the electrical circuit.
 
Start/Stop Button

The Start/Stop Button is located near the blotting surface and is used to activate the run, stop the run, or reset the program. The red and green status light indicates the status of the run or errors.
 
Control Panel

The Control Panel is located near the de-bubbling surface and contains the 6-digit digital display, Select button, and Up/Down (+/-) Buttons.
 
Programs

The iBlot™ Gel Transfer Device is pre-programmed with 5 voltage programs that allow blotting using a different combination of volts and time for each gel type. The 5 programs are listed in the table below.

Program
Volt
Default Run Time
Run Time Limit
P1
25
6 minutes
10 minutes
P2
23
6 minutes
11 minutes
P3
20
6 minutes
13 minutes
P4
15
6 minutes
16 minutes
P5
10
6 minutes
25 minutes


The Default Run Time is the default time shown for each program which can be increased or decreased using the Up/Down (+/-) Buttons. The Run Time Limit is the maximum run time that can be programmed for the specific program.  Select an appropriate program based on the gel type that you wish to blot.
 
 
iBlot™ Anode Stack, Bottom

The iBlot™ Anode Stack, Bottom package contains a copper electrode, nitrocellulose (0.2 µm) membrane, and the Bottom Transfer Gel Layer packaged in a transparent plastic tray. The transparent plastic tray serves as the support for assembling the transfer stacks with the gel and has a tab that assists in the movement of the transfer stack assembly towards the blotting surface during the de-bubbling process. The Bottom Transfer Gel Layer acts as an ion reservoir and is composed of an optimized, proprietary gel composition to provide high-quality transfer of proteins within 7 minutes.

The nitrocellulose (0.2 µm) membrane does not require any pre-treatment before use and minimizes protein blow-through during the iBlot™ blotting process.

Always use the iBlot™ Anode Stack, Bottom with the tray in the iBlot™ Device.
See details on iBlot™ Cathode Stack specifications.




The iBlot™ Anode Stack, Bottom is available in standard format for blotting E-PAGE™, midi, or two mini gels and Mini format for blotting one mini gel.
Dispose the iBlot™ Anode Stack, Bottom after every use. Do not reuse the iBlot™ Anode Stack, Bottom.
 
 
iBlot™ Cathode Stack, Top

The iBlot™ Cathode Stack, Top package contains a copper electrode and the Top Transfer Gel Layer packaged in a red, plastic tray. The Top Transfer Gel Layer acts as an ion reservoir and is composed of an optimized, proprietary gel composition to provide high-quality transfer within 7 minutes.
See iBlot™ Cathode Stack specifications




The iBlot™ Cathode Stack is available in standard format for blotting E-PAGE™, midi, or two mini gels and Mini format for blotting one mini gel.

Dispose the iBlot™ Cathode Stack, Top after every use. Do not reuse the iBlot™ Cathode Stack, Top. Do not use the iBlot™ Cathode Stack, Top with the tray in the iBlot™ Device.
 
iBlot™ Disposable Sponge

The iBlot™ Disposable Sponge is placed on the inner side of the iBlot™ Lid within the small protrusions on the lid. The iBlot™ Disposable Sponge absorbs any excess liquid on the stacks formed during blotting and generates an even pressure on the stack assembly. See dimensions of the iBlot™ Disposable Sponge.




The iBlot™ Disposable Sponge is comprised of a blue, polystyrene sponge, a white felt, and a brass metal contact. The metal contact is fixed onto the sponge at a distance of 15 mm from the upper right corner. The metal contact allows proper contact with the electrical contact on the lid as well as the electrode on the assembled iBlot™ Gel Transfer Stacks.

Discard the iBlot™ Disposable Sponge after every use. Do not reuse the iBlot™ Disposable Sponge.

 
iBlot™ Filter Paper

The iBlot™ Filter Paper is used for blotting mini or midi gels. The iBlot™ Filter Paper is placed on top of the thin gel before placing the iBlot™ Cathode Stack, Top to protect the gel integrity during the blotting process. The iBlot™ Filter Paper is supplied in two sizes  for efficient blotting of mini and midi gels. The iBlot™ Filter Paper is not used for blotting E-PAGE™ gels.

Note: Failure to use the iBlot™ Filter Paper during blotting of mini or midi gels may result in high currents exceeding the current limit leading to an error (Error2) during the run.
 
iBlot™ E-PAGE™ Tab

The iBlot™ E-PAGE™ Tab is a steel tab used during blotting of E-PAGE™ gels. The iBlot™ E-PAGE™ Tab is attached to the iBlot™ Cathode Stack, Top and is used to pull the transfer stack assembly towards the blotting surface during the de-bubbling process of E-PAGE™ gels.
 
De-Bubbling Roller

The De-bubbling Roller is a stainless steel, aluminum roller designed to remove any air bubbles between the gel and blotting membrane during the assembly of the stacks and gel for blotting E-PAGE™ gels. The De-bubbling Roller is installed into the hinges on the de-bubbling surface. The iBlot™ Gel Transfer Stacks and gel are aligned between the Metal Spacers 1 and 2, and the De-bubbling Roller is placed on top. The entire gel assembly is pulled together with the pull tab towards the blotting surface to efficiently remove any air bubbles. Use the protocol  to perform blotting using the De-Bubbling Roller. Do not use the De-bubbling Roller for mini, midi, or gels other than E-PAGE™ gels as these gels may stretch and tear.




Blotting Roller

The Blotting Roller is a Delrin roller attached to a stainless steel handle (8.6 cm wide). The Blotting Roller is used to remove any air bubbles between the gel and blotting membrane during the assembly of the stacks and gel. Use the protocol to perform blotting of gels using the Blotting Roller.



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Experimental Outline

The table below outlines the experimental steps necessary to perform Western blotting using the iBlot™ Gel Transfer Device.

Step
Action
1
Remove the gel from the gel cassette.
2
Assemble the iBlot Gel Transfer Device with the iBlot Gel Transfer Stacks and your protein gel using:
  • De-bubbling Roller
  • Blotting Roller
3
Perform Western blotting using the recommended parameters.
4
Disassemble the iBlot Gel Transfer Device.


Materials Needed

You will need the following items.

  • iBlot™ Transfer Stack for blotting E-PAGE™, Novex® Midi Gels, or two mini gels (see recommended gel types)
  • iBlot™ Transfer Stacks, Mini for blotting one mini gel
  • Pre-run gel containing protein samples and protein standards

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General Guidelines

Introduction

General guidelines for using the iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stacks are discussed below.
 
Recommended Gel Types

The gel types compatible for use with iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stacks are listed below.
Note: The iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stack is not yet optimized for Northern or Southern blotting with agarose gels (E-Gel®) or TBE polyacrylamide gels.


 

Gel Type
Size
iBlot Gel Transfer Stack
E-PAGE 48 or 96 Gels
13.5 cm (l) x 10.8 cm (w)
3.7 mm thick
iBlot Gel Transfer Stack, Regular
Midi Gels
(NuPAGE® Novex® Bis-Tris, Tris-Acetate, or Tris-Glycine Midi Gels, or equivalent gels)
13 cm (l) x 8.3 cm (w)
1.0 mm thick
iBlot Gel Transfer Stack, Regular
Mini Gels
(NuPAGE® Bis-Tris or Tris-Acetate, Tricine, Tris-Glycine Gels or equivalent gels)
8 cm (l) x 8 cm (w)
1.0 and 1.5 mm thick
iBlot Gel Transfer Stack, Regular or iBlot Gel Transfer Stack, Mini



Recommended Parameters

Use the following parameters for blotting based on the gel type that you use.

Gel Type Program Volts Run Time
E-PAGE™ 48P2237-8 minutes
E-PAGE™ 96P2237-8 minutes
Novex® Midi Gel, 1 mm thickP2236 minutes
2 Mini Gels (1.0 or 1.5 mm thick)P2236 minutes
1 Mini Gel (1.0 or 1.5 mm thick) using mini transfer stacksP2236 minutes



Custom parameters are also easily created using a combination of programs (P1-P5) and time (up to the time limit listed for each program) for a gel types not listed above.
 
 
Recommended Protocols

Use the following recommended blotting protocols based on the gel type that you use:

  • For E-PAGE™ gels, use the blotting protocol with the De-bubbling Roller as described
  • For mini or midi gels, use the blotting protocol with the Blotting Roller as described 


Recommendations

To obtain the best results, follow these recommendations:

  • Wear gloves at all times during the entire blotting procedure to prevent contamination of gels and membranes.
  • Do not touch the membrane or gel with bare or gloved hands. This may contaminate the gel or membrane and interfere with further analysis. If you need to adjust the membrane, always use forceps.
  • Use the appropriate gel type and iBlot™ Gel Transfer Stacks as described.
  • Avoid using expired iBlot™ Gel Transfer Stacks. Always use the transfer stacks before the specified expiration date printed on the package.
  • Remove air bubbles as indicated in the protocol using the De-bubbler Roller or Blotting Roller supplied with the device.
  • Do not trim the membrane or iBlot™ Gel Transfer Stacks to fit your gel size. See above for the gel sizes that are compatible with iBlot™ Device. Note that iBlot™ Gel Transfer Stacks, Mini are available for blotting mini gels.


We have

Introduction

General guidelines for using the iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stacks are discussed below.
 
Recommended Gel Types

The gel types compatible for use with iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stacks are listed below.
Note: The iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stack is not yet optimized for Northern or Southern blotting with agarose gels (E-Gel®) or TBE polyacrylamide gels.


 

Gel Type
Size
iBlot Gel Transfer Stack
E-PAGE 48 or 96 Gels
13.5 cm (l) x 10.8 cm (w)
3.7 mm thick
iBlot Gel Transfer Stack, Regular
Midi Gels
(NuPAGE® Novex® Bis-Tris, Tris-Acetate, or Tris-Glycine Midi Gels, or equivalent gels)
13 cm (l) x 8.3 cm (w)
1.0 mm thick
iBlot Gel Transfer Stack, Regular
Mini Gels
(NuPAGE® Bis-Tris or Tris-Acetate, Tricine, Tris-Glycine Gels or equivalent gels)
8 cm (l) x 8 cm (w)
1.0 and 1.5 mm thick
iBlot Gel Transfer Stack, Regular or iBlot Gel Transfer Stack, Mini



Recommended Parameters

Use the following parameters for blotting based on the gel type that you use.

Gel Type Program Volts Run Time
E-PAGE™ 48P2237-8 minutes
E-PAGE™ 96P2237-8 minutes
Novex® Midi Gel, 1 mm thickP2236 minutes
2 Mini Gels (1.0 or 1.5 mm thick)P2236 minutes
1 Mini Gel (1.0 or 1.5 mm thick) using mini transfer stacksP2236 minutes



Custom parameters are also easily created using a combination of programs (P1-P5) and time (up to the time limit listed for each program) for a gel types not listed above.
 
 
Recommended Protocols

Use the following recommended blotting protocols based on the gel type that you use:

  • For E-PAGE™ gels, use the blotting protocol with the De-bubbling Roller as described
  • For mini or midi gels, use the blotting protocol with the Blotting Roller as described 


Recommendations

To obtain the best results, follow these recommendations:

  • Wear gloves at all times during the entire blotting procedure to prevent contamination of gels and membranes.
  • Do not touch the membrane or gel with bare or gloved hands. This may contaminate the gel or membrane and interfere with further analysis. If you need to adjust the membrane, always use forceps.
  • Use the appropriate gel type and iBlot™ Gel Transfer Stacks as described.
  • Avoid using expired iBlot™ Gel Transfer Stacks. Always use the transfer stacks before the specified expiration date printed on the package.
  • Remove air bubbles as indicated in the protocol using the De-bubbler Roller or Blotting Roller supplied with the device.
  • Do not trim the membrane or iBlot™ Gel Transfer Stacks to fit your gel size. See above for the gel sizes that are compatible with iBlot™ Device. Note that iBlot™ Gel Transfer Stacks, Mini are available for blotting mini gels.


We have observed increased immunodetection sensitivity with the iBlot® 7-Minute Blotting System. If you are using the iBlot™ system for the first time, you may need to load less protein, use more diluted antibody for detection, or perform detection for a shorter time as compared to traditional semi-dry or wet blotting systems. You may need to optimize the immunodetection based on your initial results.  If you are using the iBlot™ system for the first time, you may need to load less protein, use more diluted antibody for detection, or perform detection for a shorter time as compared to traditional semi-dry or wet blotting systems. You may need to optimize the immunodetection based on your initial results.

 

 


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Getting Started

Installing the iBlot™ Gel Transfer Device

  1. Check the Power Cord supplied with the unit to ensure that the cord is compatible with the local socket format.

  2. Place the iBlot™ Gel Transfer Device on a level laboratory bench. Keep the area around the device clear to ensure proper ventilation of the unit.

  3. For your safety: Position the device properly such that the power switch and the AC inlet located on the rear of the unit are easily accessible.

  4. Ensure the AC power switch is in the Off position.

  5. Attach the power cord to the AC inlet and then to the electrical outlet. Use only properly grounded AC outlets and power cords.
 
You are ready to use the iBlot™ Gel Transfer Device for blotting applications. See the blotting procedure.
 
Using the iBlot™ Device for the First Time

If you are using the iBlot™ Gel Transfer Device for the first time, you may wish to clean the Metal Spacers 1 and 2, De-bubbling Roller, and blotting surface with a damp cloth before use. Allow the parts to dry before blotting.
 
Selecting a Program

You will need to select an appropriate program on the iBlot™ Device prior to assembling the device with iBlot™ Gel Transfer Stacks and your gel.

  1. When the electrophoresis of your samples is almost complete, press the power switch (located on the rear of the device) to turn ON the iBlot™ Gel Transfer Device.  The fan in the device begins to run and digital display shows text which is stabilized in few seconds to display the default parameters (P 1.0 7:00) or last program used.

  2. Select the appropriate program based on the gel type by pressing the Select button to toggle between program, minutes, and seconds. Once the selected item blinks, use the Up/Down (+/-) Buttons for changing the values to the desired parameters as shown below:
 
Gel Type Program Volts Run Time
E-PAGE™ 48 P2237-8 minutes
E-PAGE™ 96P2237-8 minutes
Novex® Midi Gel, 1 mm thick P2236 minutes
2 Mini Gels (1.0 or 1.5 mm thick)P2 236 minutes
1 Mini Gel (1.0 or 1.5 mm thick) using mini transfer stacksP2 236 minutes

Custom parameters are also easily created using a combination of programs (P1-P5) and time (up to the time limit listed for each gel type) for a specific gel type not listed above.

Note:  You may need to optimize the blotting parameters (volts or time) based on your initial results. See details on optimizing blotting conditions.
 
The maximum voltage and current of the output to gel stacks is 25 VDC and 5.5 Amp

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Using the iBlot™ Device with the De-Bubbling Roller

Introduction

Instructions are provided in this section to assemble the iBlot™ Gel Transfer Device with the De-Bubbling Roller for blotting E-PAGE™ Gels.
If you wish to blot mini, midi, or other gels, see details on the blotting protocol.
 
Materials Needed

You will need the following items:

  • Pre-run E-PAGE™ gel or equivalent containing your protein samples and standards
  • iBlot™ Gel Transfer Stacks

 
Removing the Gel

Remove the gel from the cassette for transfer after completion of electrophoresis as described below.
Open the E-PAGE™ cassette using the red plastic Butterfly Opener supplied with the gel to remove the E-PAGE™ gel. For details, refer to the E-PAGE™ manual supplied with the gel.
 
Note

There is no need for any pretreatment of the gel after electrophoresis. Wash the E-PAGE™ gel briefly in deionized water to remove any small gel pieces attached to the gel.

To use other blotting membrane instead of the nitrocellulose included with the transfer stacks:

  1. Wet the desired blotting membrane in deionized water (for PVDF membrane, wet the PVDF membrane in methanol and rinse in deionized water).

  2. Remove the nitrocellulose membrane from the stacks using forceps.

  3. Place the wet blotting membrane on the transfer stack, align the membrane flush to the stack, and remove any air bubbles using the blotting roller.


To obtain the best blotting results with the E-PAGE™ gels, we recommend that you use the De-bubbling Roller. However, you may use the Blotting Roller for de-bubbling E-PAGE™ gels as described.
 
Assembling the iBlot™ Device

Instructions are provided below to assemble the iBlot™ Gel Transfer Device with iBlot™ Gel Transfer Stacks and E-PAGE™ pre-cast gels.

  1. Open the lid of the device and pull up the Metal Spacers 1 and 2. If you have attached the De-bubbling Roller to the device, then remove the roller as shown in the figure below.



  2.  
  3. Remove the package labeled iBlot™ Anode Stack, Bottom from the iBlot™ Gel Transfer Stacks Box. Remove the laminated sealing of the iBlot™ Anode Stack, Bottom and keep the stack in the transparent plastic tray.




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  4. Place the iBlot™ Anode Stack, Bottom stack with the tray to the left of the blotting surface area such that the tab on the tray is on the right side of the De-bubbling Roller, as shown below. Slide the bottom stack to the left until the stack is blocked by the Gel Barriers present on the left side of the device. Note:   Always handle the iBlot™ Anode Stack, Bottom using the plastic tray without disturbing the gel and membrane layers in the stack. Do not touch the nitrocellulose membrane on the stack. If you wish to replace the nitrocellulose membrane with any other blotting membrane, see Note.



  5. Clean the Metal Spacer 1 with a damp cloth or tissue and place the spacer on the membrane as shown below.


  6. Place the pre-run gel containing your protein samples on Metal Spacer 1 such that the gel is aligned to the lower right corner of the bottom stack with the wells of the E-PAGE™ gel facing up.



  7. Clean the Metal Spacer 2 with a damp cloth or tissue and place the spacer over the gel as shown below.


  8. Remove the package labeled iBlot™ Cathode Stack, Top from the iBlot™ Gel Transfer Stacks Box. Remove the iBlot™ Cathode Stack, Top from the package.

  9. Insert the steel iBlot™ E-PAGE™ Tab in the plastic tray groove with the tab teeth facing up (figure A). Gently press the iBlot™ Cathode Stack over the teeth to allow the teeth to penetrate into the copper electrode (figure B). Remove the iBlot™ Cathode Stack, Top from the red plastic tray using the iBlot™ E-PAGE™ Tab (figure C).



     

  10. Place the iBlot™ Cathode Stack, Top without the tray on top of Metal Spacer 2 with the copper electrode side facing up. Ensure that all layers are aligned to the right to perform efficient de-bubbling.

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  11. Insert the De-bubbling Roller into the two grooves and lower the roller to its lowest location while holding the pull tab. The resulting assembly consists of the gel, cathode and anode stacks placed between two Metal Spacers 1 and 2 with the De-bubbling Roller on top of the assembly as shown below.


  12. Hold the iBlot™ E-PAGE™ Tab and plastic tab on the iBlot™ Anode Stack, Bottom together and pull the assembly (anode and cathode stacks, and gel) together through the De-bubbling Roller towards the blotting surface, in one smooth, uninterrupted movement until the assembly reaches the Gel Barriers on the blotting surface (figure A). At the end of de-bubbling, all layers are aligned to the right as shown below (figure B)


  13. Place the iBlot™ Disposable Sponge on the inner side of the lid (between the small protrusions on the lid that hold the sponge in its place) such that the white side is facing you and metal contact is to the top right.






The sponge absorbs any excess liquid generated during blotting and exerts an even pressure on the stack surface.
 
Performing Blotting

After assembling the iBlot™ Gel Transfer Device, perform blotting as described below. Perform blotting within 15 minutes of assembling the stacks with the gel.

  1. Close the iBlot™ Lid and secure the latch. The red light is on indicating a closed circuit. Ensure the correct program is selected. Select the appropriate program.

  2. Press the Start/Stop button to start the transfer. The red status light changes to green. The transfer continues using the parameters that you programmed as described.

  3. The iBlot™ Gel Transfer Device signals the end of transfer with repeated beeping sounds, and flashing green light and digital display.

  4. Press and release the Start/Stop button to stop the beeping. The light turns to a steady red light.

  5. Proceed to Disassembling the iBlot™ Gel Transfer Device.

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Using the iBlot™ Device with the Blotting Roller

Introduction

Instructions are provided in this section to assemble the iBlot™ Gel Transfer Device without the De-Bubbling Roller for blotting mini, midi, or other gels.
If you wish to blot E-PAGE™ gels, see for the blotting protocol.
 
Materials Needed

You will need the following items:

  • Pre-run mini or midi gel containing your protein samples and standards
  • iBlot™ Gel Transfer Stacks for blotting one midi gel or two mini-gels
  • iBlot™ Gel Transfer Stacks, Mini for blotting one mini gel
  • Blotting Roller supplied with the device

 
Removing the Gel

Remove the gel from the cassette for transfer after completion of electrophoresis as described below.

  • Open the mini or midi gel cassette using the Gel Knife by inserting the knife into the narrow gap between the two plates of the cassette. Push up and down gently on the knife’s handle to separate the plates. Upon opening the cassette, discard the plate without the gel and slowly remove the gel adhered to the other plate. For details on removing the gel, refer to the manual supplied with the mini or midi gel.
  • For other gel types, refer to the manufacturer’s recommendations to remove the gel from the cassette


Note

There is no need for any pretreatment of the gel after electrophoresis.

You may blot E-PAGE™ gels using the blotting protocol with the Blotting Roller. If you wish to use the Blotting Roller for blotting E-PAGE™ gels be sure to:

  • Wash the E-PAGE™ gel briefly in deionized water prior to blotting to remove any small gel pieces attached to the gel.
  • Use the Blotting Roller all over the gel including all well areas to obtain efficient blotting.


If you notice distorted protein bands after using the E-PAGE™ blotting protocol with the Blotting Roller, we recommend that you blot the E-PAGE™ gels using the De-bubbling Roller.
 
To use other blotting membranes instead of the nitrocellulose included with the transfer stacks:

  1. Wet the desired blotting membrane in deionized water (for PVDF membrane, wet the PVDF membrane in methanol and rinse in deionized water).

  2. Remove the nitrocellulose membrane from the stacks using forceps.

  3. Place the wet blotting membrane on the transfer stack, align the membrane flush to the stack, and remove any air bubbles using the blotting roller.

 
Use the appropriate iBlot™ Gel Transfer Stacks based on the gel that you are blotting.  Do not trim the membrane or transfer stacks to fit the size of your gel. Use the iBlot™ Gel Transfer Stacks, Mini for blotting one mini gel.
 
 
Assembling the iBlot™ Device

Instructions are provided below to assemble the iBlot™ Gel Transfer Device with iBlot™ Gel Transfer Stacks or Mini, and mini, midi, or other gels. See details on blotting E-PAGE™ gels.

  1. Open the lid of the iBlot™ Gel Transfer Device. Ensure the blotting surface is clean.

  2. Remove the iBlot™ Anode Stack, Bottom (or Mini stack) from the package. Remove the laminated sealing of the iBlot™ Anode Stack, Bottom and keep the stack in the transparent plastic tray. Place the iBlot™ Anode Stack, Bottom with the tray directly on the blotting surface (under the round lid). Align the anode stack to the Gel barriers on the blotting surface (see figure below).




  3.  
  4. Ensure no bubbles are visible between the nitrocellulose membrane and the transfer stack gel below the membrane. Remove any trapped air bubbles using the Blotting Roller. Place the pre-run gel on the nitrocellulose membrane of the anode stack as described:

  5. One midi gel on an iBlot™ Gel Transfer Stack Two mini gels (head-to-head) on an iBlot™ Gel Transfer Stack (figure A) One mini gel on an iBlot™ Gel Transfer Stack, Mini (figure B).


  6. In a clean container, soak one iBlot™ Filter Paper (or Mini Filter paper based on the gel type used) in deionized water. iBlot™ Filter Paper is included with each iBlot™ Gel Transfer Stacks. Place the pre-soaked iBlot™ Filter Paper on the pre-run gel. Use the Blotting Roller to remove any air bubbles between the membrane and gel as shown below for the Transfer Stack. For E-PAGE™ gels, there is no need to use a filter paper and be sure to use the Blotting Roller over the well rows to flatten any remaining gel protrusions to ensure even transfer.

  7. Remove the iBlot™ Cathode Stack, Top (or Cathode Stack, Mini) from the package. Discard the red plastic tray. Place the iBlot™ Cathode Stack, Top (or Cathode Stack, Mini) on top of the pre-soaked filter paper with the copper electrode side facing up and aligned to the right of the bottom stack. Remove any air-bubbles using the Blotting Roller

  8. Place the iBlot™ Disposable Sponge on the inner side of the lid (between the small protrusions on the lid that hold the sponge in its place) such that the white side is facing you and metal contact is to the top right as shown below for the Mini Transfer Stack.



The sponge absorbs any excess liquid generated during blotting and exerts an even pressure on the stack surface.
 
Performing Blotting

After assembling the iBlot™ Gel Transfer Device, perform blotting as described below. Perform blotting within 15 minutes of assembling the stacks with the gel.

  1. Close the iBlot™ Lid and secure the latch. The red light is on indicating a closed circuit. Ensure that the correct program is selected. Select the appropriate program.

  2. Press the Start/Stop button to start the transfer. The red status light changes to green. The transfer continues using the parameters that you programmed as described.

  3. The iBlot™ Gel Transfer Device signals the end of transfer with repeated beeping sounds and flashing green light and digital display.

  4. Press and release the Start/Stop button to stop the beeping. The light turns to a steady red light.

  5. Proceed to Disassembling the iBlot™ Gel Transfer Device

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iBlot™ Quick Reference Guide

Mode
Action
Sound
Light
Display
iBlot plugged in
iBlot connected to an electrical outlet and power switch is on
-- --
Version of iBlot firmware
iBlot turned on
No transfer stacks detected
with lid opened
-- --
Default setting or the last defined program/time appear
Program selection
Press and release the Select Button
-- --
Program name blinks
Time selection
(minutes)
Press the Select Button and use the Up and Down Buttons (+/-) to change values
-- --
Minutes blink
Time selection
(seconds)
Press the Select Button and use the Up and Down Buttons (+/-) to change values
-- --
Seconds blink
Ready to run
Transfer stacks placed in the device and lid closed
--
Steady red
Program and time
Run
Press and release the Start/Stop button
--
Steady green
Count down time
Running error alert
Open the lid and fix the error (lost contact with stacks or short circuit)
Continuous
beeping
Flashing red
Error1 or Error2
Error fixed
Close the lid
Continuous
beeping
Flashing green
Error1 or Error2
Continue after error
Press and release the Start/Stop button
--
Steady green
Count down time
Restart after error
Press and hold the Start/Stop button
  • Transfer stacks placed in the device and lid closed

 

  • No transfer stacks detected with lid opened
--
 
  Steady red
 
 
 
 
 
 
 
          
 
 
  • Program and time

 

  • Default setting or the last defined program/time appear
End of run
Automatic
Continuous beeping for 2 minutes (or less if Start/Stop button is pressed) followed by a single beep every minute
Flashing green
Program and time
Run ends after an external power failure
Transfer stacks placed in the device and lid closed
             --
Steady red
Program and time

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Disassembling the iBlot™ Gel Transfer Device

Introduction

Refer to the instructions below to disassemble the iBlot™ Gel Transfer Device.
 
Procedure

To obtain good transfer and detection results, disassemble the device and stacks within 30 minutes of ending the blotting procedure.

  1. Open the lid of the iBlot™ Device.

  2. Remove the iBlot™ E-PAGE™ Tab (used for blotting E-PAGE™ gels only). Rinse the tab with deionized water and store in a dry place for future use. Do not discard the iBlot™ E-PAGE™ Tab.

  3. Discard the iBlot™ Disposable Sponge and iBlot™ Cathode Stack, Top.

  4. Carefully remove and discard the gel and filter paper (if used) as shown below. Remove the nitrocellulose membrane from the stack and proceed with the blocking procedure or stain the  membrane.




  5. Discard the iBlot™ Anode stack, Bottom.

  6. At this point, the iBlot™ Gel Transfer Device is ready for another run (no cooling period is required). If you are not using the device, turn off the power switch located on the back of the iBlot™ Gel Transfer Device.


 
Do not reuse the iBlot™ Disposable Sponge, iBlot™ Filter Paper, and iBlot™ Cathode and Anode Stacks after blotting. Discard after each use.
 
 
Cleaning and Maintenance

Clean the blotting surface, Metal Spacers 1 and 2, and the De-bubbling Roller with a damp cloth or paper tissue. Allow the parts to dry before use.
For any other repairs and service, contact Technical Service. Do not perform any repairs or service on the iBlot™ Gel Transfer device to avoid damaging the iBlot™ Device.


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Post Transfer Analysis and Optimizing Blotting

Post Transfer Analysis

After the transfer, you may proceed to immunodetection, store the membrane for future use, or stain the membrane.

  • For immunodetection of proteins, use the WesternBreeze® Chromogenic or Chemiluminescent Immunodetection Kits available from Invitrogen or any other immunodetection kit.
  • For storing nitrocellulose membranes, air dry the membrane and store the membrane in an air-tight plastic bag at room temperature or 4°C. Avoid storing nitrocellulose below -20°C, as they turn brittle.
  • For staining membranes after blotting, you may use any total protein membrane staining methods such as Coomassie® Blue R-250, Ponceau S, Amido Black, or SYPRO® Ruby Blot Stain. The iBlot™ Gel Transfer Device blotting protocol is compatible with most total protein membrane staining methods listed above.

Note:  The sensitivity of total protein membrane staining after using the dry blotting protocol with iBlot™ Gel Transfer Device is slightly lower than the total membrane protein staining obtained with the semi-wet transfer protocol.
 
If you do not detect any proteins on the membrane after immunodetection or staining, refer to Troubleshooting.  Refer to the manufacturer’s recommendations for optimizing immunodetection.
 
Optimizing Blotting

When using the iBlot™ Gel 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.
Optimization of blotting can be performed as follows:

  • Increasing or decreasing the transfer time.  Based on the initial results, you can increase or decrease the transfer time using the Up/Down buttons. Do not perform transfer for more than the time limit indicated for each program.
  • Performing an equilibration step prior to transfer.  To improve the transfer of high-molecular weight proteins from mini or midi gels, equilibrate the gel in 100 ml Equilibration Buffer (2X NuPAGE® Transfer Buffer containing 10% methanol and 1:1000 NuPAGE® Antioxidant) for 20 minutes at room temperature on a shaker prior to transfer. After the equilibration step, use the gel for transfer using the iBlot™ Gel Transfer Device as described in this manual.

Note:  The equilibration step improves the transfer of mini or midi NuPAGE® or Tris-Glycine gels only. Do not use this equilibration step with E-PAGE™ gels as no improvement in the transfer is observed.
 
It is normal for some proteins to remain in the gel as some high molecular weight proteins do not transfer completely using the iBlot™ Gel Transfer Device as compared to semi-wet transfer apparatus. Since the sensitivity of detection using the iBlot™ Gel Transfer Device is much higher as compared to semi-wet and semi-dry blotting, complete transfer of proteins is not required. Almost complete transfer of pre-stained standard protein bands is observed with the iBlot™ Gel Transfer Device. However, note that the complete transfer of pre-stained protein standards does not indicate complete transfer of other proteins.

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Examples of Results

Introduction

Examples of results obtained after blotting mini and E-PAGE™ gels using the iBlot® 7-Minute Blotting System as shown below.
 
E-PAGE™ Gel Results

E-PAGE™ 48 8% Gel was subjected to blotting after electrophoresis using the iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stacks with the De-bubbling Roller as described in this manual. The proteins on the nitrocellulose membrane were detected using the WesternBreeze® Chemiluminescent Anti- Mouse Kit using 1:10,000 dilution of anti-BSA antibody (left panel) or 1:10,000 dilution of anti-tubulin antibody (right panel).
The gel contains the following samples (rows not indicated are blank):

Lane      
Sample
2, 3, 4, 5, 6, and 26, 27, 28, 29, 30        
BSA (5 ng, 10 ng, 25 ng, 50 ng, and 100 ng)
 8, 9, 10, 11, and 14, 15, 16, 17
MagicMark XP Western Protein
32, 33, 34, 35, and 38, 39, 40, 41
Standard (0.5 µl, 1 µl, 2 µl, and 4 µl)
19, 20, 21, 22, 23, and 43, 44, 45, 46
Human Colon Cancer cell lysate, SW480 (0.25 µl, 0.5 µl, 1 µl, 2 µl, and 4 µl)







Mini Gel Results

Two NuPAGE® Novex® 4-12% Bis-Tris Gels were subjected to blotting after electrophoresis using the iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stacks with the Blotting Roller, as described in this manual. The proteins on the nitrocellulose membrane were detected using the WesternBreeze® Chromogenic Anti-Rabbit Kit using 1:2,000 dilution of an anti-E. coli antibody.
Samples on the gel:
Lane 1:  5 µl SeeBlue® Plus2 Pre-Stained Protein Standard
Lanes 2-9:  Duplicate samples of E. coli lysate diluted 1:16 (0.5 µl, 1 µl, 2 µl, 4 µl, respectively)






Mini Gel Results, continued

SeeBlue® Plus2 Pre-Stained Protein Standard (5 µl) was electrophoresed on a NuPAGE® Novex® 4-12% Bis-Tris Gel. After electrophoresis, the gel was subjected to blotting using the iBlot™ Gel Transfer Device and iBlot™ Gel Transfer Stacks, Mini with Blotting Roller as described in this manual.
The figure below shows good transfer of protein standard bands on to the nitrocellulose membrane.


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Troubleshooting

Problem Cause Solution
No current (red light is not on after securing the lid)Incomplete electric circuit due to:

iBlot™ Disposable Sponge covers the metal contact or the metal contact on the sponge is on the left

Incorrect position of the transfer stacks or improper assembly of the transfer stacks

 

Incorrect position of the pull tab

 

iBlot™ Anode Stack, Bottom placed on the device without the tray including the electrical contact

 

iBlot™ Cathode Stack, Top placed on the device with the tray

Reinsert the iBlot™ Disposable Sponge such that the metal contact on the sponge is on the top right of the lid and is in contact with the electrode on the transfer stack.

Make sure the transfer stack is placed in the proper position in the blotting surface to allow proper contacts with the electrodes.

Ensure the transfer stacks are assembled correctly; use the iBlot™ Anode Stack, Bottom first followed by the gel and iBlot™ Cathode Stack, Top.

Ensure the pull tab from the iBlot™ Cathode Stack, Top is towards the right of the assembly in the blotting surface.

Do not remove the iBlot™ Anode Stack, Bottom from the tray during the assembly. The blotting is performed with the bottom stack in the plastic tray.

Always remove the iBlot™ Cathode Stack, Top from the red plastic tray before placing the top stack on the assembly. Do not use the iBlot™ Cathode Stack, Top with the tray.

Digital display shows Error1 indicating an open electrical circuit during the runThe lid opened during the runClose the lid. Continue the run by briefly pressing Start/Stop button or restart the run by pressing and holding the Start/Stop button.
Digital display shows Error2 indicating a short circuitThe iBlot™ Cathode Stack, Top is touching the copper electrode on the iBlot™ Anode Stack, Bottom

The layers are not aligned


Current is above 5.5 amp

Open the lid and align the iBlot™ Cathode Stack, Top to the right. Continue the run by briefly pressing Start/Stop button or restart the run by pressing and holding the Start/Stop button.

Align the layers properly as described in the protocols. Ensure that the electrodes are in contact.

Select a program with a lower voltage. Replace the iBlot™ Gel Transfer Stacks with fresh transfer stacks. Ensure that the iBlot™ Filter Paper was used during blotting of mini or midi gels.

Corrosion of the iBlot™ Cathode Stack, TopIncorrect placement of the top stackBe sure the iBlot™ Cathode Stack, Top is placed correctly with the copper electrode side facing up. Avoid placing the top stack in the inverted position.
Excessive liquid noticed on the iBlot™ Disposable Sponge or the iBlot™ Anode Stack, Bottom is filled with liquidiBlot™ Disposable Sponge placed incorrectlyAlways place the iBlot™ Disposable Sponge into the lid with the white side facing the user. Do not place the sponge into the lid with the blue side facing the user.
No proteins transferred to the membrane No current or incorrect program used See to ensure the electrical circuit is complete and current is flowing through the device. Be sure to use the appropriate program based on the gel type.
Empty spots on the membranePresence of air bubbles between the gel and the membrane preventing the transfer of proteins

Expired or creased membranes used

Be sure to remove all air bubbles between the gel and membrane by using the De-Bubbling Roller for E?PAGE™ Gels or Blotting Roller for other gels.

Use the iBlot™ Gel Transfer Stacks before the expiration date printed on the package.

Protein bands distorted on membrane (for E-PAGE™ gels)Non-uniform electric field created around wellsEnsure that the well protrusions on the E-PAGE™ gel are flattened properly using the De-bubbling Roller or the Blotting Roller.
To ensure best blotting results, we recommend using the De-bubbling Roller with E-PAGE™ gels. If you used the Blotting Roller with E-PAGE™ gels, be sure to follow the recommendations to obtain good results.
Proteins remain in the gel indicated by staining of the gel after transferIncorrect program usedUse the appropriate program and run time based on the gel type as described.
You may perform an equilibration step with Equilibration Solution as described to improve transfer.
It is normal for some proteins to remain in the gel as some proteins do not transfer completely using the iBlot™ Gel Transfer Device as compared to semi-wet transfer apparatus.
Membrane and the gel turns blueLonger transfer times result in the deposition of copper ionsBe sure to perform the transfer for the recommended time for each gel type.
Signal intensity is similar for different protein loads after detectionHigh protein load (detection is not within the linear range)Since the immunodetection sensitivity is higher for dry blotting with iBlot™ device when compared to semi-dry or wet blotting, we recommend that you decrease the protein load, use more diluted antibody, or perform detection for shorter time. You may need to perform some optimization based on your initial results.

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25-0911  MAN0000560    21-Dec-2009