Mini Transblot And Transblot Systems From Bio-Rad (for mini and maxi gels, respectively)

Western blotting, a technique which is the “bread and butter” for the cancer biologist requires an efficient, reliable apparatus for the transfer of important protein/DNA samples which are sometimes rare (e.g. human tissues). Depending on the requirements, either a wet or dry transfer is used for protein transfer onto membrane. In our lab, wet transfer is suitable because we are investigating a large range of proteins, including high molecular proteins (40 Kd – 350 Kd). We have transblot apparatus from different companies like Biotech, Bangalore genei, Bio-Rad and even apparatuses made by our own institute’s workshop. Among all of these, the Transblot from Bio-Rad is the best.

The Mini-Transblot (7.5 x 10 cm blotting area) and Transblot (16 x 20 cm blotting area) from Bio-Rad consists of tank, tank lid with permanently attached cords, a pair of cassettes, filter paper and fiber pads. They have color-coded electrophoretic blotting cells and the cassettes. Color-coded here is an important feature which helps in setting the correct orientation of gel (i.e. gel on the black side and membrane on the white/red side). The Mini-Transblot requires 1 L of transfer buffer and Transblot requires 3 L of buffer. The Mini-Transblot cell contains a Bio-Ice cooling unit which helps maintain the temperature during transfer at a constant 100V for 1 hour. However, we never use the cooling unit; instead, we use chilled buffer and perform the transfer in front of an air conditioner. The Mini-Transblot comes with platinum wires. The Transblot cell (16 x 20 cm) consists of platinum-coated titanium and stainless steel plate electrodes. The set also contains a supercooling coil (which we do not use as we never perform high intensity transfers). We have found the plate electrodes perform optimally and have never thought of shifting to platinum wire electrodes. Transblot electro transfer is carried out at constant 55 Volts for 3 hours in chilled buffer and in a cold room with constant stirring. Also, filter papers supplied by Bio-Rad are very convenient. They immediately absorb water without accumulating any air bubbles. This was not the case with our Whatman paper which we used previously for our transfers.

For blotting, the black side of the cassette is placed in buffer in a tray followed by a fiber pad and then the gel is transferred on the dry filter paper and is placed on the black side of the cassette. After activation of PVDF membrane, it is placed onto the gel and filter paper is placed onto the membrane followed by another fiber pad. For avoiding the air bubbles, we roll a test tube on the filter paper before placing the fiber pad. The cassette can be closed very easily. The complete cassette is then placed in the tank which is then filled with buffer.

I perform blotting for both one and two dimensional electrophoresis. For one dimension gel, the issue is not a major one as normal and tumor tissues can be transferred on one membrane by loading them in two different wells. However in case of 2DGE, this does become an issue if 2 different gels are transferred in different cassettes. I have found that transfer is not 100% efficient in both the cassettes of the same apparatus at the same time; there is some variation in protein transfer. When comparing the 2D Western blot and when working with an increase or decrease of a specific protein, one has to have both the gels on the same membrane to rule out the error of unequal transfer. For this purpose, the Transblot has proved to be excellent. I transfer 8-12 gels at a time, keeping 2 normal and 2 tumor 2DGE gels (total 4 gels) on a single membrane: 4 mini gels fit in one single cassette of the Transblot. However, it is a little more tedious as one has to fit 4 gels in the correct orientation and mark them as normal and tumor. One has to be very alert as there is a risk of breaking the gel while transferring onto the filter paper. Also, the membrane is to be cut properly as extra membrane generates more heat. However, in the end, this apparatus gives the clearest results. We also use the Transblot for identification of different keratins in one dimension. There are 2-8 different keratins expressed by every epithelial cell. Their molecular weights fall in the range of 40-70 Kd. Hence, loading the tissue lysate on a 16 cm gel gives better separation, which we transfer with the Transblot.

In summary, I would recommend these Transblot apparatuses to all the basic biochemistry labs, as I have found these apparatuses to be simple, reliable, durable and efficient in the transfer of proteins.