The Hoefer Semi-Dry Tranfer Unit does exactly what you think it would – it transfers proteins from SDS-PAGE gels onto membranes (either nitrocellulose or PVDF) rapidly and efficiently. It comes in two-sizes: the smaller model, the TE70, has an effective transfer surface of 14x16 cm, while the larger model, the TE77, has a transfer area of 21x26 cm. In my hands, protein transfer through this semi-dry apparatus is more efficient, economical and significantly faster (1hour vs. 3-4 hours or even overnight) than any conventional wet-transfer modules that I have tried.
The operation of this unit is pretty straightforward. Basically, the membrane is sandwiched between blotting paper which is then placed on top of the metal transfer surface (which is actually the anode of the unit). The cover is then placed on the unit (the cathode). It is called a “semi-dry” transfer unit because very little transfer buffer is required for the whole process - just enough to soak the blotting paper and membrane. For a single, 14 x 16 cm gel, 250ml of buffer suffices. The manufacturer recommends at least six pieces of blot paper on each side, but I’ve found that one piece of standard blotting paper on each side is sufficient. The only critical point to remember is to drive out any trapped air-bubbles in the membrane sandwich with a roller (I fashioned a home-made roller by cutting off the two ends of a 10-ml plastic pipette). The manufacturer also recommends that the membrane should be slightly smaller than the gel to avoid the current bypassing the gel, however, I regularly use membranes that are significantly larger than the gel, but I still wind up with perfect transfers (as judged by the complete transfer of pre-stained markers from the gel onto the membrane). Obviously the product is actually more forgiving than the manufacturer gives it credit for. After the blotting, the membrane can be stained with Ponceau-Red to confirm that the protein transfer was successful before proceeding to the next step.
With the TE77 model, the transfer is carried out with a constant current of 230 mA. For most proteins in a 1mm-thick gel, the transfer should be complete within one hour. For 1.5mm-thick gels, I usually extend the transfer to 1.5 hrs. Proteins larger than 250 kD may not get a quantitative transfer even after prolonged blotting. Two-types of transfer buffer are recommended due to their low-ionic strength (hence they produce less heat): Towbin buffer and CAPS buffer. For large membrane proteins, the Towbin buffer might be better since it contains SDS, which facilitates the transfer of hydrophobic proteins. I always use PVDF membranes since, in my hands, they tend to retain protein much better than nitrocellulose and show minimal blotting-through (over-transfer). This is especially critical when transferring both large and small proteins on the same blot. Additionally, PVDF membranes can be stripped more evenly and reproducibly than nitrocellulose. However, keep in mind that Ponceau-Red stains nitrocellulose-bound proteins much better than PVDF-bound proteins.
Besides its fast and economical nature, another major advantage of the Hoefer Semi-Dry Transfer Unit is its ability to allow the blotting of multiple gels at the same time. This is usually accomplished by stacking multiple sandwiches on top of each other. According to the manufacturer, a maximum of 3 gels can be simultaneously blotted. However, in my experience, a side-by-side parallel transfer of multiple gels offers a more consistent and comparable transfer than a vertical stacking; however, if stacking is imperative, always leave the most important gel at the bottom since the gel closest to the anode gets the best transfer.
In summary, I find the Hoefer Semi-Dry Transfer Unit to be a highly effective and indispensable protein blotting apparatus. It has significantly reduced my daily workload and facilitated my overall research.
Zhan Xiao
Postdoctoral Fellow
Whitehead Institute for Biomedical Research
Cambridge, MA