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iBlot™ 3 Western Blot Transfer Device, 1 device - FAQs

iBlot™ 3 Western Blot Transfer Device, 1 device - FAQs

View additional product information for iBlot™ 3 Western Blot Transfer Device - FAQs (A56727, IB31001)

34 product FAQs found

I used PVDF Gel Transfer Stacks with the iBlot 3 Western Blot Transfer System, and my proteins did not transfer to the membrane. What happened?

This is likely due to the PVDF membrane being dry or partially dry. Regions where PVDF membranes are dry appear whiter than places where the membrane is wet. Remove the membrane and reactivate in 100% methanol, and rinse in water before reapplying to the transfer stack.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

After transferring using the iBlot 3 Western Blot Transfer System, I noticed that the Bottom Stack transfer gel melted to a viscous blue solution. Why is this?

This happens if the membrane is trimmed to fit the gel size resulting in direct contact between the Top and Bottom transfer stacks. This can be avoided by maintaining the membrane size identical to the transfer stack. Transfer quality is not affected by smaller gel size compared to the membrane.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

What is the Blotting Roller that is provided with the iBlot 3 Western Blot Transfer System?

The Blotting Roller is a plastic roller attached to a stainless steel handle. It is used to remove any air bubbles between the gel and blotting membrane during the assembly of the transfer stacks and gel.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Can I cut the membrane in the iBlot 3 Transfer Stacks to fit my separating gel?

No, we recommend maintaining the membrane size identical to the transfer stacks. This helps ensure that there is no direct contact between the Top and Bottom transfer stacks.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Are the PVDF and nitrocellulose membranes in the iBlot 3 Transfer Stacks compatible with fluorescent antibodies or membrane staining (e.g., LI-COR, Ponceau S)?

The PVDF and nitrocellulose membranes in the iBlot 3 Transfer Stacks are compatible with all commonly used detection methods such as staining, immunodetection, fluorescence, etc.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Can I replace the membrane in the transfer stack with my preferred membrane when using the iBlot 3 Western Blot System?

Yes, it is possible to replace the membrane provided in your transfer stack with any membrane that is compatible with western blotting. To do this, cut the alternative membrane to match the size of your gel, and wet the membrane. Then either place the alternative membrane on top of the integrated membrane, or carefully remove the integrated membrane from the gel matrix with forceps and replace it with the new membrane. Note that Thermo Fisher Scientific only supports the use of transfer stacks when they are used with the provided instructions.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Are the copper electrodes in the iBlot 3 Transfer Stacks recyclable?

To maximize the recovery of the copper from the mesh electrodes used in the iBlot 3 Transfer Stacks, we have established an agreement with a recycling center in the United States. In order to prepare the copper electrodes for recycling, please follow the instructions listed here.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

I want to perform western transfer with mini gels (8 x 8 cm), but I do not have iBlot 3 Transfer Stacks (Mini). Can I use iBlot 3 Transfer Stacks (Regular) to transfer my mini gels?

It is best not to transfer a single mini gel on a regular-sized transfer stack. Although in most cases, the transfer will be fine, empty spaces on the transfer stack that are not in direct contact with the gel could potentially cause distortions across the whole surface of the membrane, including the portion in contact with the gel. It is best to have more than 50% of a membrane in contact with the gel, if possible. Our recommendation is that you transfer 2 mini gels using a Regular transfer stack.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

In the iBlot 3 Top Stack, I see a white sheet beneath the transfer gel layer. What purpose does it serve?

This is the plastic separator that separates the Top Stack from the Bottom Stack. Right before assembling the transfer stack, this separator needs to be removed from the Top Stack and discarded.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Can I use the iBlot 3 Transfer Stacks after the expiration date?

No, we do not recommend using the iBlot 3 Transfer Stacks after the expiration date. Use of transfer stacks after the expiration date can lead to inferior results.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

What are the different kinds of iBlot 3 Transfer Stacks that you offer and are they available separately?

We offer iBlot 3 Transfer Stacks with integrated nitrocellulose or PVDF membranes for transferring proteins from gels onto nitrocellulose or PVDF membranes, respectively (western blotting). These transfer stacks are available in Midi and Mini format and can also be purchased separate from the iBlot 3 Western Blot Transfer Device.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Can I use the iBlot 3 Transfer Stacks and/or iBlot 3 Absorbent Pad more than once?

No, the iBlot 3 Transfer Stacks contain a finite number of ions to drive the transfer and are depleted after a single use. We also recommend discarding the iBlot 3 Absorbent Pad after every use.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Is there a limitation on the thickness of gels that can be used with the iBlot 3 Western Blot Transfer Device?

The iBlot 3 Western Blot Transfer Device is compatible with gels with a thickness of ≤1.5 mm.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Are the iBlot 3 Transfer Stacks compatible with the original iBlot and iBlot 2 transfer devices?

Unfortunately, the iBlot 3 Transfer Stacks are not compatible with the original iBlot and iBlot 2 transfer devices. The iBlot 3 Transfer Stacks are only compatible with the iBlot 3 Western Blot Transfer Device.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Are the E-PAGE Gels (example Cat. No. EP09606) compatible with the iBlot3 device (Cat. O. IB31001)?

Due to the thickness of the E-PAGE gels, they are not compatible with the iBlot3 device for transfer. Recommended methods for transfer of E-PAGE gels are semi-dry blotting or semi-wet blotting in the XCell II Blot Module. Methods are described in the E-PAGE Technical Guide.

Find additional tips, troubleshooting help, and resources within our Protein Gel Electrophoresis Chambers, Power Supplies, and Accessories Support Center.

Do you offer additional years of service for the iBlot 3 Western Transfer Device?

No, we do not provide service contracts or extended warranties for these devices. The warranty is limited to 1 year from the date of shipment.

With the iBlot 3 Western Blot Transfer System, some of my proteins blew through the membrane. What could have caused this?

This could happen if the transfer time used is too long. We recommend reducing the transfer time by 30 sec increments.
Note: Pre-stained markers are charged, so they tend to blow-through more than regular proteins.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

How can I get better transfer of high-molecular weight proteins using the iBlot 3 Western Blot Transfer System?

Proteins larger than ~150 kDa migrate more slowly than smaller proteins. Therefore, more time is required to transfer them from the gel to the membrane. We recommend using a transfer time of 8-10 mins for optimal transfer of these proteins.

To enhance transfer efficiency, we also recommend adding a 5 min water wash step between electrophoresis and western transfer and using a Tris-Acetate gel for protein separation. For the protocol, please refer to page 36 in the iBlot 3 Western Blot Transfer System user manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0028464-iBlot3WesternTransferSys-UG-EN.pdf).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Can the iBlot 3 Western Blot Transfer System be used for transfer of low- and high-molecular weight proteins?

Yes. However, minor optimizations in the transfer protocol (i.e., steps, voltage, time, and cooling) may be needed for some proteins. Also, gel type is an important consideration. More details and suggestions for optimizing the transfer protocol can be found on page 36 of the iBlot 3 Western Blot Transfer System user manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0028464-iBlot3WesternTransferSys-UG-EN.pdf).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

What are the different kinds of iBlot 3 Transfer Stacks that you offer and are they available separately?

We offer iBlot 3 Western Blot Transfer Stacks (mini and midi formats) with integrated nitrocellulose or PVDF membranes.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

What kind of gels can I use with the iBlot 3 Western Blot Transfer System for western blotting?

The iBlot 3 Western Blot Transfer System is compatible with Bis-Tris, Tris-Acetate, Tris-Glycine, and Tricine (in mini- and midi-gel formats). Please note that the iBlot 3 Western Blot Transfer system is not compatible with E-PAGE gels.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

I see a lot of empty spots on the membrane after transferring using the iBlot 3 Western Blot Transfer System. What could have caused this?

Here are possible causes and solutions:

- Presence of air bubbles between the gel and the membrane preventing the transfer of proteins. Be sure to remove all air bubbles between the gel and membrane using the Blotting Roller.
- Expired or creased membranes used. Use the iBlot 3 Transfer Stacks before the expiration date printed on the package.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

I used the iBlot 3 Western Blot Transfer System and none of my proteins transferred to the membrane. What could have happened?

This can happen if the stack was not assembled correctly or if two mini stacks were transferred simultaneously in the same station and at least one of the stacks was not assembled properly. We recommend checking to make sure that stacks are assembled correctly. A schematic of the iBlot 3 Transfer Stack showing the flow of current can be found on page 10 of the user manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0028464-iBlot3WesternTransferSys-UG-EN.pdf).

This problem can also occur if the incorrect method and/or gel type was used. Please check to make sure that the correct method and gel type were used. A list of recommended transfer conditions and gel types can be found on pages 20 and 23 of the user manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0028464-iBlot3WesternTransferSys-UG-EN.pdf), respectively.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

After transferring using the iBlot 3 Western Blot Transfer System, both my membrane and the gel turned blue or green. Is this normal?

Longer transfer times result in the deposition of copper ions. Be sure to perform the transfer for the recommended time for each gel type. This can also occur if the stack is not disassembled within 5 mins of completing the transfer.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Do you offer replacement electrodes for the iBlot 3 Western Blot Transfer System?

Yes, we offer the iBlot 3 Western Blot Transfer System Electrode Replacement Kit, Cat No. A56427. Please refer to pages 39-40 in the iBlot 3 Western Blot Transfer System user manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0028464-iBlot3WesternTransferSys-UG-EN.pdf) for instructions to replace the electrodes.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

What methods come pre-programmed in the iBlot 3 Western Blot Transfer System?

The iBlot 3 Western Blot Transfer System comes pre-programmed with the following three methods:
- Broad Range (30-250 kDa): 25 V for 6 mins with low cooling
- High Molecular Weight (150-400 kDa): 30 V for 8 mins with no cooling
- Low Molecular Weight (10-120 kDa): 25 V for 3 mins with medium cooling

Please note that custom methods can also be created. Instructions for creating custom methods can be found on pages 28-31 of the iBlot 3 Western Blot Transfer System user manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0028464-iBlot3WesternTransferSys-UG-EN.pdf).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

What is the purpose of the built-in cooling feature in the iBlot 3 Western Blot Transfer System?

The built-in cooling feature allows for improved transfer consistency with the iBlot 3 Western Blot Transfer System by reducing temperature build-up. We recommend using the cooling feature when performing back-to-back transfers to keep the system cool and/or when transferring low molecular weight proteins (<30 kDa) to prevent the proteins from blowing through the membrane.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

How many gels can I transfer simultaneously using the iBlot 3 Western Blot Transfer System?

Each independently controlled station of the iBlot 3 Western Blot Transfer System can transfer 1 or 2 mini gels simultaneously, or 1 midi gel.

If both stations of the iBlot 3 Western Blot Transfer System are utilized, then up to 4 mini gels or 2 midi gels can be transferred simultaneously.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.