One of the most common techniques in modern biological laboratories is Western blotting. The technique requires the electrophoretic transfer of proteins that have been electrophoretically separated on a polyacrylamide gel to nitrocellulose or other types of membrane. The transfer can be performed either in wet (tank) or semi-dry conditions. The Bio-Rad Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell is a very efficient, highly reproducible blotting system, especially for proteins with low molecular weight.
The unique design allows protein transfer in a horizontal configuration without a buffer tank or gel cassettes. The anode platform moves up and down to accommodate various gel stack thicknesses, producing even pressure on the blotting sandwich during transfer. Moreover, direct contact between the plate electrodes and filter paper stack leads to more efficient and faster transfers. With a semi-dry system, transfer can be done in 15–30 minutes for mini gels and 30–60 minutes for full-size gels, compared to a few hours to overnight with the traditional tank system. The Bio-Rad Trans-Blot® Semi-Dry System has a large surface area (20 x 18.5 cm) for blotting and allows the transfer of up to 3 small gels and 2 large gels at a time.
The transfer protocol is rather simple. After the proteins are separated by electrophoresis on a polyacrylamide gel, the gel and pieces of nitrocellulose membrane and filter paper are cut to the size of the gel and then wetted and equilibrated in transfer buffer for about 15 minutes. Next, the transfer sandwich is prepared starting from the bottom platinum anode with 2-3 pieces of filter paper, then the membrane, the equilibrated gel and finally another 2-3 pieces of filter paper. After the sandwich is arranged, I usually roll a glass cylinder across it to eliminate air bubbles. I then secure the stainless steel cathode and the safety cover. Then the assembly is clamped and plugged into the power supply. The transfer can be run at different voltages and for different lengths of time. I usually transfer mini gels at 15 V for 15 minutes, and larger gels at 25 V for 30 min. After the transfer, the membrane is placed in a Ponceau solution and the markers are drawn with a pencil. Next the membrane is used for classic Western blot procedures.
To get a more efficient transfer, one should incubate the gel and membrane in transfer buffer before the transfer and make sure there are no air bubbles or extra buffer trapped between the gel and membrane by rolling a glass pipette over the sandwich a few times. Also, assembling the transfer sandwich with paper, gel and membrane of the same size reduces the area exposed to the electrode and thus, the current required for transfer; this minimizes local heating effects. Other variables that influence the uniformity of the transfer are the size of the protein, percentage of gel used, strength of the electric field and the time of the transfer. With the semi-dry system, some low molecular weight molecules will not transfer quantitatively, while molecules greater than 110 kD sometimes do not transfer at all.
The minimal amount of buffer required for the transfer is very small as buffer is required only to wet the membrane and the filter paper. In contrast, tank systems require up to 1 liter of buffer per transfer.
The single-step locking system on the Trans-Blot® SD Cell is very simple and the entire apparatus requires very little maintenance: it only needs to be wiped off with 70% ethanol after use. The only parts that are less durable are the screws that keep the safety cover in place; these fell off after intensive use over the past 3 years.