iBlot™ DNA Transfer Stack - FAQs

Yes, all original iBlot stacks are still available for purchase. You can find them in the original iBlot Gel Transfer Device Reagents and Resources (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/iblot-dry-blotting-system/reagents-resources-original-iblot-gel-transfer-device.html). However, please note that they are not compatible with the iBlot 2 Gel transfer Device.

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

No. Do not use iBlot Transfer Stacks in the iBlot 2 Gel Transfer Device, and do not mix components between iBlot Transfer Stacks and iBlot 2 Transfer Stacks.

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

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 to 10 minutes for optimal transfer of proteins >150 kDa using the iBlot Dry Blotting System.

To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE Invitrogen 3 - 8% Tris-acetate gels for electrophoresis. We have an application note available, titled “Transferring Large and Small Proteins Using the iBlot Dry Blotting System”. To download the PDF, see the iBlot Reagents Resources page. (http://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/western-blotting/western-blot-transfer/iblot-dry-blotting-system/original-iblot-resources.html)

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

This may be a result of insufficient blocking or nonspecific binding. We suggest trying our WesternBreeze Blocker/Diluent (Cat. No. WB7050). 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.

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

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 we only support the use of iBlot 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.

The minimum guaranteed shelf life of iBlot Transfer Stacks is 2 months. Depending on when you purchase the transfer stack, shelf life will be 2–8 months. The expiration date is printed on the package for each stack.

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

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.

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).

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

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.

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

The plastic in the iBlot Dry Blotting System stack packaging is polyethylene terephthalate (PET) and can be recycled.

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

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.

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