Blotting equipment has become an invaluable tool in modern protein biochemistry. At this point there are two main types of transfer methods – conventional, tank-style electroblotting and semi-dry electroblotting. Each work by the same basic principle; namely moving proteins from the gel matrix to a membrane by electromotive force. The tank-style apparatus immerses the gel-membrane sandwich in a tank of buffer to which an electric field is applied. The semi-dry method places the gel-membrane sandwich in between two large opposing electrodes using blotting paper that has been soaked in buffer. Due to the small gap between the electrodes, a low voltage electrical field with a high current can be used to move the proteins out of the gel and onto the membrane. This type of setup reduces that amount of buffer used and because of the high electrical field strength reduces the time needed for transfer.
The Panthertm Semidry Electroblotter made by Owl Scientific is a simple and very reliable transfer apparatus. It consists of two electrodes, one platinum/titanium and the other stainless steel, each measuring 20cm by 20cm set into a low profile acrylic case (Owl also produces a 35cm by 45cm Panther™ to accommodate larger gels). The unit functions by placing one electrode in the base and the other in the lid, thus the transfer occurs in a horizontal configuration. The unit is secured together by nylon-threaded bolts matted to acrylic/brass knobs.
Unfortunately producing reliable results with any transfer apparatus requires attention to detail and a fair amount of practice. Thankfully using the Panther™ unit is straightforward and not complicated. Many different buffers recipes are provided in the instruction manual, each suited for different applications. This unit can accommodate both protein as well as nucleic acid transfers. I have found that a standard Tris-base (25mM), glycine (192mM), methonal (20%) buffer is adequate for transfers of proteins up to 80kDa. For larger proteins, I include SDS (1%) to improve the transfer. I must also remark that with a semi-dry apparatus the gel, membrane and blotting paper should be cut to the same size to ensure a uniform transfer. In contrast to the tank-transfer system, the semi-dry system is run at constant current, rather than a constant voltage, giving a higher field strength and shorter transfer times. I currently use 1.5mm thick gels and find that 2 hours is more than sufficient to transfer proteins smaller then 80kDa. For larger proteins I would increase the transfer time to three hours. Because the membrane area changes for different applications, I use the formula of 1mA per square centimeter of total membrane area when calculating the proper current for each transfer. This allows multiple gels to be transferred at the same time, in a side-by-side configuration. Due to the high field strength I have noticed that if the transfers are performed with the gel/membrane sandwiches one on top of another, proteins can move from one gel through the membrane into the gel above resulting in artifacts. For this reason adjusting the transfer times is critical.
The only negatives that I have found with this unit are the nylon bolts. After many uses they can become stripped and make it almost impossible to get the knob to fit. I was able to get replacement parts from Owl that have the bolts already matted to the knobs. This configuration works much better and is less prone to cross threading and stripping of the nylon threads. This is the only problem that I have had with this unit in the 3 years I have used it. Others in my lab have used a water filled 4 L flask as a weight to secure the lid to the base instead of the knobs, without changing their results.
In general I have found the Panther™ unit to be a very solid performer. It is straightforward to use and is made to provide consistent results.
Eric Deszo, PhD Student
University of Illinois Champaign-Urbana
Department of Animal Sciences