iBlot® 7-Minute Blotting System FAQs

Below are some frequently asked questions and answers regarding the iBlot® 7-Minute Blotting System and iBlot® Western Detections Kits. If you have a question that is not listed below or need additional information, please contact our Technical Support department.

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iBlot® Frequently Asked Questions
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Dry Blotting Device Technical Questions

Is there a limitation on the thickness of gels that can be used with the iBlot® 7-Minute Blotting System?
The iBlot® 7-Minute Blotting System has been tested to efficiently transfer protein from gels ranging in thickness from 1 mm to 3 mm. We have not tested gels thicker than 3 mm because they are rarely used for SDS-PAGE.

I want to conduct western transfer with mini gels (8 x 8 cm), but I don’t have iBlot® Transfer Stacks (Mini). Can I use iBlot® 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.

How can we be more environmentally responsible?
The plastic in the iBlot® 7-Minute Blotting System stack packaging is polyethylene terephthalate (PET) and can be recycled.

Are the copper electrodes in the transfer stacks recyclable?
No. The electrodes are copper-coated nylon, and the amount of copper left in the electrode after a transfer is rather small. There is 0.6 grams of copper per sheet before transfer, and even less after transfer.

Sometimes there is green discoloration on the blot around the gel. What is this, and does it affect the results?
The green discoloration is copper deposits from the iBlot® Transfer Stacks, 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.

Do the PVDF iBlot® Transfer Stacks (0.2 µm, non-autofluorescing) require activation prior to use?
No! The PVDF membrane comes preactivated. You just need to open the transfer stack with membrane, place the separation gel on top of the membrane, and apply one layer of moistened filter paper to run (the same as with the nitrocellulose stacks).

Can iBlot® Transfer Stacks be used more than once?
No. The transfer stacks have a finite amount of ions to drive the transfer and are depleted after a single use.

What is the shelf life of both the nitrocellulose and PVDF iBlot® Transfer Stacks?
The minimum guaranteed shelf life for iBlot® Transfer Stacks is 2 months. Depending on when you purchase the transfer stack, shelf life will be 2–8 months.

Is it possible to substitute the membrane from the iBlot® Transfer Stack with my specialized membrane?
In theory, you can replace the membrane provided in your iBlot® 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 Life Technologies only supports the use of iBlot® Transfer Stacks when they are used with the provided instructions.

 

General Dry Blotting Technical & Application Questions

Occasionally my western blots have high background. What do you recommend?
This may be a result of insufficient blocking or nonspecific binding. We suggest trying our WesternBreeze® Blocker/Diluent. We have been using it with good success. Additionally, you should optimize primary and secondary antibody concentrations as generally recommended for any new blotting technique. Many cases of high background can be resolved by further diluting one or both antibody preparations.

How can I get better transfer of high molecular weight proteins?
Proteins larger than ~150 kDa migrate more slowly than smaller proteins. Therefore, more time is required to transfer them from gel to membrane. We recommend extending the transfer time by 8–10 minutes for optimal transfer of proteins >150 kDa using the iBlot® 7-Minute Blotting System.

To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE® Novex 3-8% Tris-acetate gels for electrophoresis. We have an application note available, titled “Transferring Large and Small Proteins Using the iBlot® 7-Minute Blotting System”. To download the file, see the iBlot® Technical Resources page.

What causes empty spots on my membrane after transfer?
The iBlot® 7-Minute Blotting System is similar to conventional transfer methods in that air bubbles between the gel and the membrane will prevent protein transfer. Be sure to remove all air bubbles between the gel and membrane before starting the transfer, using the blotting roller supplied with the iBlot® 7-Minute Blotting System.

Is there a stripping protocol for the iBlot® 7-Minute Blotting System?
conventional stripping protocol using 0.1 M glycine, pH 2, works with polyclonal antibodies.

Does the iBlot® 7-Minute Blotting System work with native or native-blue gels?
Yes, we have an application note available, titled, “Western Blotting NativePAGE™ Novex® Bis-Tris gels Using the iBlot® 7-Minute Blotting System’. To download the file, see the iBlot® Technical Resources page.

Does the iBlot® 7-Minute Blotting System work with WesternDot™ (Qdot® 625 nanocrystal) western kits?
This application is currently being investigated.

 

Questions About iBlot® Western Detection Kits

Do I need to have an iBlot® 7-Minute Blotting System to use an iBlot® Western Detection Kit?
Yes. The iBlot® Western Detection Kits use the iBlot® system to produce an electrical field that accelerates interactions between antibodies and blocking reagents with membrane-bound antigens. iBlot® Western Detection Kits require an iBlot® 7-Minute Blotting System with the P9 program, found in current firmware version 2.9.5. If you have an older iBlot® Dry Blotting System, the firmware is freely available for download. See the Instrument Registration page to find out how.

Can iBlot® Western Detection Stacks be used more than once?
No. The detection stacks have a finite amount of ions and are depleted after a single use, which includes the three detection steps: blocking, primary antibody, and secondary antibody.

Should I change the detection stacks for each step?
No. A single set of stacks is used for all three steps of the western detection.

Does it matter what method I use to transfer protein to my western blot membrane?
No, it does not matter. iBlot® Western Detection Kits are compatible with western blots created using wet or semi-dry methods. You can use one regular-sized membrane (13.5 cm x 8 cm) or 1–2 mini-sized membranes (8 x 8 cm).

How many reactions can I perform at one time?
iBlot® Western Detection Stacks come in two sizes: mini and regular; they are supplied with assay spacers to create partitions for analysis using more than one set of antibodies. With the mini size you can perform detection on one mini-sized membrane or two halves of a mini-sized membrane. With the regular size you can perform detection on two mini-sized membranes or 4 quarters of a regular-sized membrane (4 x 8 cm).

What is the sensitivity of the iBlot® Western Detection Kits?
The iBlot® Western Detection Kits offer comparable or better sensitivity than conventional protocols for chemiluminescent or chromogenic detection (Figure 1).

Comparison of iBlot® Western Detection Kit to WesternBreeze® kit
Figure 1. Comparison of iBlot® Western Detection Kit to WesternBreeze® kit. Proteins from an SW480 (human colon adenocarcinoma cell line) lysate were transferred using the iBlot® 7-Minute Blotting System. Mouse anti-p53 antibody was used as the primary antibody. Detection was performed with (A) the WesternBreeze® Chemiluminescent Kit—Anti-Mouse or (B) the iBlot® Western Detection Chemiluminescent Kit (Anti-Mouse). The iBlot® Western Detection Kit detected the proteins with sensitivity comparable to the WesternBreeze® kit.

Sometimes I see high background on the membrane after detection. How can I eliminate it?
High background can be caused by nonspecific antibody binding. To eliminate it, use a lower concentration of the secondary antibody (as specified in the manual). In addition, using nitrocellulose rather than PVDF membranes will help to minimize background.

Sometimes my molecular weight (MW) markers are no longer visible after my detection is completed. Does this mean my proteins are also gone?
Definitely not. MW markers are usually prestained, and the stains are more highly charged than typical cellular proteins. Applying an electrical field in the western detection procedure can drive the MW markers right through the membrane onto the bottom stack.

Cellular proteins, however, are less charged than the MW markers, and at the detection stage they no longer contain any SDS. The result is that the cellular proteins stay immobilized on the membrane during western detection using the iBlot® system.

Can I use different antibodies or different dilutions of the same antibody in the procedure?
Yes. iBlot® Western Detection Kits are supplied with reusable spacers, expressly for this purpose. Use spacers to separate membrane sections for detection with different antibody solutions at the same time. A detailed protocol is provided in the user manual.

What membrane type should I use with the iBlot® Western Detection Kits? Is one type preferred over another?
You can use nitrocellulose or PVDF membranes with the iBlot® Western Detection Kits. However, we highly recommend using nitrocellulose rather than PVDF because less background is seen with nitrocellulose membranes. This results in more sensitive detection.

Can I use the iBlot® Western Detection Kits on membranes with previously transferred proteins?
Yes, as long as you activate the membrane before you start the detection procedure. Completely wet nitrocellulose membranes using water. Activate PVDF membranes by immersing in 100% methanol for several seconds until the membrane is completely wet, then rinse with water. Then proceed with the iBlot® Western Detection Kit.

What substrate is used with the kits?
The substrate used for the iBlot® Western Detection Kits reacts with alkaline phosphatase (AP). Therefore, horseradish peroxidase (HRP) substrates will not work for this kit. Other conjugates and substrates of AP cannot be used with iBlot® Western Detection Kits. The reagents supplied with the kits have been optimized to obtain the best results.

How is it possible to finish the entire immunodetection in just 30 minutes?
Native antibodies typically have some negative charge at neutral pH, which allows their electrophoretic migration in low voltage from the carrier matrix onto the membrane. The force of the electric field is counterbalanced by their affinity for antigen, so that binding antibodies are “captured” while unbound antibodies flow through the blocked membrane and onto the bottom stack. Basically, the iBlot® system electrophoretically focuses antibodies to greatly accelerate their interaction rate with immobilized antigens.

How can I improve the signal intensity?
The signal intensity is primarily controlled by the concentration of the secondary antibody; nonspecific binding can result when the concentration is too high, and poor binding to the primary antibody can result when the solution is too dilute. The next option is to modify the primary antibody concentration, which controls the number of antibody molecules that bind to the antigen, and thus the signal and background levels. (See the manual for further details.)

Are the kits compatible with the LiCor system?
Currently, the iBlot® Western Detection Kits use a green matrix, and unfortunately this can create background fluorescence that isn’t compatible with the LiCor Odyssey scanner. White matrices with low or no fluorescence will be included in the next version of the iBlot® Western Detection Kits. We anticipate release of the LiCor-compatible iBlot® Western Detection Kit in 2011. Please contact your local Life Technologies sales representative for information.

Can the primary antibody solution be reused?
No. The primary antibody solution cannot be reused after it has been applied to the matrix.

What concentration of primary antibody should I use with the iBlot® Western Detection Kits?
Use twice the primary antibody concentration used in typical western blot detection procedures. Note that the overall amount of the primary antibody used is the same as in typical traditional methods, because only half the volume of primary antibody solution is needed with the iBlot® western detection system.

What is the shelf life of the iBlot® Western Detection Stacks?
The shelf life is 6 months, at room temperature.

What is the shelf life of the iBlot® Western Detection Reagents?
The shelf life is 12 months, at 4°C.

I have reagents left but the detection stacks have expired. Can I purchase only the iBlot® Western Detection Stacks?
Yes. To use your reagents with new stacks, you can purchase the stacks only: iBlot® Western Detection Stacks, regular size and iBlot® Western Detection Stacks, mini size.

Is there a website with further details?
Yes. For more information, go to our page about iBlot Western Detection Kits.

Is there a demo video to explain the procedure and protocol?
For a demonstration video, see our page about iBlot Western Detection Kits. The video is also available for viewing on YouTube.

 

For Research Use Only.  Not intended for any animal or human therapeutic or diagnostic use.