Trans-Blot® Electrophoretic Transfer Cell From Bio-Rad

Conventional Southern, Northern and Western blotting are methods are based on the specific binding of either complementary nucleic acid strands (in the case of DNA and RNA) or specific antigen-antibody binding (in the case of proteins) in order to detect the particular oligonucleotide or protein of interest. These bio-molecules are first separated by agarose or polyacrylamide gel electrophoresis and then transferred from the gel on to a solid support (i.e. membrane). This transfer can be achieved with the aid of capillary pressure, vacuum force or an electric field. Capillary and vacuum blotting methods are generally employed for the transfer of nucleic acids; however, electro-blotting is suitable for most of types of transfers (protein and nucleic acid). Electro-blotting can be done semi-dry or wet, in the case of proteins. Semi-dry electro-blotting is not as efficient as wet transfer for high molecular weight proteins.

Our lab has been using trans-blot apparatuses (one made in our workshop and one commercially available) for a long time. However, we have found the Trans-Blot® Cell from Bio-Rad (cat #170-3939) to be suitable for our protein and nucleic acid transfers. The Trans-Blot® Electrophoretic Transfer Cell facilitates wet transfers of up to three 16 x 20 cm gels or up to twelve mini format gels, simultaneously. The dimensions are 24 x 18 x 9.5 cm and it requires 3 L of buffer. The Trans-Blot® Cell is available with durable plate electrodes or more economical wire electrodes. We perform electroblotting using the plate electrodes. The electrodes may be positioned 4 cm or 8 cm apart and this flexibility, along with a variable power setting, permits standard field (30 V), overnight transfers or high-intensity field (200 V) transfers for rapid, hour-long experiments. The anode consists of platinum-coated titanium and the cathode is made of stainless steel. In addition, 2 color-coded gel holder cassettes (effective blotting area of 16 x 20 cm), a complete set of fiber pads and a super-cooling coil are provided. The super-cooling coil maintains a constant buffer temperature during non-denaturing protein, high-intensity, or overnight transfers.

For blotting, a gel sandwich is made and placed on the gray side of the cassette. Air bubbles in the sandwich have to be avoided. The cassette is closed and placed in the tank into which chilled transfer buffer is poured. We perform wet transfer for high molecular weight proteins like PCNA, HDAC1 and pRb. Also, DNA fragments separated by PAGE are transferred using TAE buffer.

We predominantly use the Trans-Blot® Cell to transfer of proteins; we perform electroblotting at 55 Volts for 3 hours with constant stirring. Maintenance of temperature is critical for efficient transfer and hence we maintain the temperature at 4ºC. We often transfer 8 mini gels at a time using the Trans-Blot® Cell. Also, this minimizes the efforts taken to transfer 8 separate minigels in 4 different mini-transblot cells, which is more tedious, and time-consuming. As we study some very high molecular weight proteins (~220 and 240 Kd), we also use maxigels for the separation of proteins and blot onto PVDF membrane. When using a on a 6% minigel, we loose low molecular weight proteins like beta-actin (42 Kd) which we use as a loading control. Hence, a 7.3% maxigel is a better option as it allows for the identification of all three proteins. One can also load more than 20 different samples on a maxigel, which gives better representation on the same blot; this is not possible with minigels. The larger format has also proven very useful in studying proteins very close in molecular weight as studying them in the minigel format is simply too difficult.

In terms of efficiency of protein transfer and for handling large amounts of work at a time, we found the Bio-Rad Trans-Blot® Cell simply great.