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iBlot™ 2 Transfer Stacks, PVDF, regular size, 10 Stacks - FAQs

iBlot™ 2 Transfer Stacks, PVDF, regular size, 10 Stacks - FAQs

View additional product information for iBlot™ 2 Transfer Stacks, PVDF, regular size - FAQs (IB24001)

38 product FAQs found

If a green color appears on the membrane after the transfer using the iBlot 2 Dry Blotting System, will it affect the results?

The green discoloration is copper deposits from the transfer stack, and it does not affect the results. To minimize this effect, shake excess water off the filter paper and buffer from the gel before placing each on the stack. The current formulation of stacks minimizes the green discoloration.

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

Is the PVDF membrane in the iBlot 2 PVDF Gel Transfer Stack pre-activated?

The PVDF membrane is pre-activated and ready for use without any pre-treatment with alcohol.

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

Can I use the stack and/or absorbent pad more than once when using the iBlot 2 Dry Blotting System?

No. The transfer stacks have a finite amount of ions to drive the transfer and are depleted after a single use.

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

Can I perform multiple transfers, with a new transfer starting immediately after the previous one when using the iBlot 2 Dry Blotting System?

Yes. The device is designed to do so with no impact on performance.

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

What types of membranes are available in the iBlot Transfer Stacks?

iBlot Transfer Stacks are available with either an integrated 0.2 µm nitrocellulose membrane or a 0.2 µm PVDF membrane. Both types are available in two sizes: a mini size which can accommodate one mini gel (8 x 8 cm), or regular size which can accommodate one E-PAGE gel, one midi gel (8 x 13 cm), or two mini gels (8 x 8 cm).

The catalog numbers are as follows:
IB3010-01 iBlot Gel Transfer Stacks, Regular (10 Blots)
IB3010-02 iBlot Gel Transfer Stacks, Mini (10 Blots)
IB4010-01 iBlot Transfer Stack PVDF, Regular (10 Blots)
IB4010-02 iBlot Transfer Stack PVDF, Mini (10 Blots)

If you would like to blot proteins onto your own membrane rather than the supplied nitrocellulose or PVDF, you can replace the integrated membrane with your desired membrane as described below:

1) Wet the desired blotting membrane in deionized water. (For PVDF membranes, first wet the PVDF membrane in 100% methanol and then rinse in deionized water).
2) Carefully remove the nitrocellulose membrane from the bottom stack using forceps.
3) Place the wetted blotting membrane on the bottom transfer stack. (Membrane should be completely wet, but not dripping; make sure there is not too much excess water.)
4) Be sure to align the membrane flush to the bottom stack gel, and remove any air bubbles using the blotting roller. Proceed with the standard iBlot transfer protocol.

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

How many gels can I transfer simultaneously using iBlot 2 Transfer Stacks?

You can transfer:
- One midi gel or two mini gels (head-to-head) using the iBlot 2 Regular Transfer Stacks
- One mini gel using iBlot 2 Mini Transfer Stacks

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

I used the iBlot 2 Dry Blotting System and noticed that the signal intensity was similar for different protein loads after detection. Why is this?

This is most likely due to the protein load being too high, as a result of which detection is not within the linear range. Since the immunodetection sensitivity is higher for dry blotting with the iBlot 2 Gel Transfer Device than for 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.

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

I used the iBlot 2 Dry Blotting System and got high background on the membrane. Can you offer some tips?

This is likely due to use of TBST buffers for washing. We recommend using PBST or WesternBreeze wash solutions.

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

I used PVDF Gel Transfer Stacks with the iBlot 2 Dry Blotting 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.

I transferred my proteins using the iBlot 2 Dry Blotting System and my protein bands are distorted on the membrane. Why is this?

This indicates that a non-uniform electric field was created around the wells. Ensure that the gel is properly flattened by using the Blotting Roller. Follow the recommendations on page 19 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf) to obtain good results.

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

With the iBlot 2 Dry Blotting 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 second increments.
Note: Pre-stained markers are charged, so tend to blow-through more than regular proteins.

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

I transferred my proteins using the iBlot 2 Dry Blotting System but the high-molecular weight proteins remained in the gel indicated by staining of the gel after transfer. Can you help me troubleshoot?

This could happen if an incorrect voltage Method was used or if inappropriate transfer conditions were used. Make sure that the voltage Method and run time used is correct, based on the gel type, as described on page 17 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf).

For mini or midi gels:
- Perform an ethanol equilibration step as described on page 35 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf) to improve transfer.
- Use a lower gel percentage to separate the high-molecular weight proteins.
- Increase the transfer time in 30-second increments.

For E-PAGE gels:
- Increase the transfer time in 30-second increments.
- Use Method P3 for 8 minutes.

Note: It is normal for some proteins to remain in the gel because some high-molecular weight proteins do not transfer completely using the iBlot 2 Gel Transfer Device, compared to semi-wet transfer apparatus.

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 transfer using the iBlot 2 Dry Blotting 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 2 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 2 Dry Blotting System and none of my proteins transferred to the membrane. What could have happened?

This could be due to no current passing through or because an incorrect voltage Method was used. Make sure that the electrical circuit is complete and current is flowing through the device. Please check to make sure that the correct voltage Method is used, see page 17 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf).

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

After transferring using the iBlot 2 Dry Blotting 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 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.

I used the iBlot 2 Dry Blotting System and noticed some green discoloration of the edges of the membrane. Will this affect the results?

The green discoloration is due to copper ions carried with liquids that get deposited onto the membrane. These deposits do not interfere with downstream processes. The stained regions can be cut away, but membrane washing typically results in their removal.

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

After transferring using the iBlot 2 Dry Blotting System, both my membrane and the gel turned blue. 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.

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

I used the iBlot 2 Dry Blotting System and noticed that the Top Stack has been corroded. What could have caused this?

This could be caused by incorrect placement of the Top Stack. Please check to make sure that the Top Stack is placed correctly with the copper electrode facing up. Avoid placing the top stack in the inverted position.

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

I am using the iBlot 2 Dry Blotting System and no current seems to be passing through. Can you help me troubleshoot?

This indicates an incomplete electric circuit. Here are possible causes and solutions:

- Incorrect placement of iBlot 2 Absorbent Pad contact: Make sure the electrical contact of the iBlot 2 Absorbent Pad is aligned with the corresponding electrical contacts on the blotting surface of the iBlot2 Gel Transfer Device.
- Top Stack placed on the device upside down: Make sure the Top Stack is assembled with the copper electrode facing up.
- The metal safety contacts in the lid hinge may be dirty and do not make contact: Clean the metal safety contacts in the lid hinge with a cotton swab and water.
- Plastic separator was not removed when assembling stack: Make sure that the plastic separator is removed from the stack, see page 21 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf).

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

What is the iBlot 2 Absorbent Pad?

The iBlot 2 Absorbent Pad absorbs any excess liquid formed on the stacks during blotting and generates even pressure on the stack assembly. It is placed on top of the assembled iBlot 2 stack prior to transfer. We recommend discarding the iBlot 2 Absorbent Pad after every use.

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 2 Dry Blotting 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 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 2 Transfer Stacks to fit my separating gel?

No. Maintain 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 iBlot 2 PVDF and nitrocellulose membranes compatible with fluorescent antibodies or membrane staining (e.g., LI-COR, Ponceau S)?

The membranes 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.

How can I get better transfer of high-molecular weight proteins using the iBlot 2 Dry Blotting 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 minutes for optimal transfer of these proteins.

To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE 3-8% Tris-acetate gels for electrophoresis. For the protocol, please refer to page 35 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblot2_device_man.pdf).

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

Can the iBlot 2 Dry Blotting 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) may be needed for some proteins.

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

Are the copper electrodes in the transfer stacks of the iBlot 2 Dry Blotting System recyclable?

To maximize the recovery of the copper mesh electrodes used in our iBlot 2 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 (https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.0358.file.dat/iBlot%202%20copper%20reycling%20instructions%20NA%20only.pdf).

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 2 Transfer Stacks (Mini). Can I use iBlot 2 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 a 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. So the recommendation would be to transfer 2 mini gels using a Regular stack.

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

Unlike the iBlot Gel Transfer Stacks, in the iBlot 2 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 stack, this separator needs to be removed from the Top Stack and discarded.

Note: In some instances, the membrane may adhere to the separator. If this is the case, use forceps to remove the membrane and place it on top of the Bottom Stack.

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

Is it okay to freeze the iBlot 2 Gel Transfer Stacks?

The iBlot 2 Gel Transfer stacks should not be frozen as they contain a gel matrix that will be damaged by freezing.

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

What are the different kinds of iBlot 2 Transfer Stacks that you offer and are they available separately?

We offer iBlot 2 Gel Transfer Stacks with integrated nitrocellulose or PVDF membranes for transferring proteins from gels onto nitrocellulose or PVDF membranes, respectively (western blotting). These stacks can also be purchased separately.

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 2 Dry Blotting System for western blotting?

The iBlot 2 Dry Blotting System is compatible for use with Bolt Bis-Tris, NuPAGE Bis-Tris, Tris-Acetate, Tris-Glycine, Tricine (in mini- and midi-gel formats), and E-PAGE gels (E-PAGE 48 only).

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

What is the purpose of the iBlot Filter Paper?

The iBlot Filter Paper is used for blotting mini- or midi-gels. It is placed on top of the gel before placing the iBlot Cathode Stack (Top Stack) 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. We do not recommend using the iBlot Filter Paper 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.

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