Invitrogen has introduced a midi-sized precast NuPAGE gel electrophoresis system and a corresponding iBlot™ system for transferring these gels. The iBlot™ system can also be used for transferring mini sized NuPAGE gels and now is available with both nitrocellulose and PVDF membranes. Our lab was one of the first to test out this system and although we have switched to almost exclusively using the NuPAGE gels for electrophoresis, we still use the NuPAGE system for transfers, rather than the iBlot™. Because of the technical issues we have encountered with the system, we only use the iBlot™ for mid-range sized proteins when quick transfers are needed.
On the positive side, the system is amazingly easy and quick to use. Our wet transfers take anywhere from 2 hours to overnight depending on the size of our protein of interest, whereas the iBlot™ only takes 7-8 minutes. It also fits midi sized gels, which we have to cut to fit into the regular NuPAGE wet transfer apparatus. The transfer stacks cost $10-12 per membrane, but you save a little bit of money by not having to make transfer buffer if you don’t have an equilibration step. You also save large amounts of time and energy when the membranes come out well. Unfortunately, the system is not reliable enough in our hands to justify using it for most of our transfers.
The iBlot™ system is described as a dry system (no buffer preparation required) for 7-8 minute transfers of protein from PAGE gels to nitrocellulose or PVDF membranes. While this is technically correct, the manual recommends an equilibration step of the gel in transfer buffer prior to iBlot™ transfer for more complete transfer of mid to high molecular weight proteins. We have found this to be a necessary step for most of our proteins of interest. This is because the transfer is known to be less complete than with a traditional wet transfer system. Although the manual also claims that the iBlot™ transfer increases sensitivity and this compensates for the less efficient transfer, we have observed decreased signal overall for mid to high range proteins in side by side comparisons to the wet system in our lab. Unfortunately, the iBlot™ only allows for 30 second adjustments to the transfer time. Thus, especially for high molecular weight proteins, an overnight wet transfer at low voltage is dramatically more efficient than with the iBlot™.
The iBlot™ also has the intermittent problem of green/brown staining of the membrane during transfer, which they say is do to buffer reactions with the copper electrode. Although this is usually around the edges only, every once in a while we have had the discoloration that extended into the area we needed to blot, and this interfered with the protein detection. There seems to be no way to tell when this will happen or a way to prevent it at this time. This is the most serious reason that we do not use the system for precious samples and reserve it for use only in cases when we absolutely need the quick transfer time.
In summary, this system is fast, easy and convenient but currently has limitations that may not make it the best choice for most labs. If you routinely transfer the same low to mid range proteins, and/or use NuPAGE midi sized gels, this may be the right choice for you. However, if you transfer a wide variety of protein sizes, high molecular weight proteins, or precious samples that are difficult to rerun, then a more reliable transfer system would be the standard NuPAGE wet transfer system.
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Rebecca M. Hall, PhD
Postdoctoral Fellow
Baylor College of Medicine
Department of Molecular & Cellular Biology
United States
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