The Trans-Blot Semi-Dry Transfer Cell manufactured by Bio-Rad incorporates the original concept of semi-dry blotting and adds new features that allow for a quicker transfer with less cleaning required. In semi-dry blotting the electrodes are placed directly in contact with the gel/nitrocellulose membrane sandwich to provide a fast, efficient transfer. Because of this direct contact there is a minimum of transfer buffer required for this process. The polyacrylamide gels must be equilibrated in transfer buffer, to remove electrophoresis buffer salts and detergents, and the nitrocellulose membranes and filter papers are also soaked, but that is all the buffer that is required (hence the term “semi-dry”). It is important to exclude excess moisture and air bubbles trapped in the filter papers and membrane when setting up the transfer, usually a pipet rolled over the surface will take care of this, but other than that, the set-up for this process is extremely simple. However, careful attention should be paid to the polarity during the set-up, since reversing the polarity can result in damage to the stainless steel cathode.
When we first began using this instrument, we spent some time determining the optimal transfer conditions for different sized gels, which took some time. In our hands, we found that we can transfer mini-gels for 15-30 minutes at 10-15 V. For large gels, the transfer takes 30 minutes to 1 hour at 15-25 V. Compared to conventional tank-transfer methods, the semi-dry method is quicker, taking a third to half the time. It should be noted that there is a current limit of 3 mA/cm2 for large gels and 5.5 mA/cm2 for mini-gels to prevent excessive heating during the transfer.
Once the transfer is complete, the efficiency can be monitored by visually checking the transfer of the pre-stained molecular weight standards. If the proteins are precipitating in the gel, the amount of SDS in the transfer buffer can be increased to enhance transfer efficiency. The draw back to this is that increasing the SDS in the transfer buffer can reduce the binding efficiency of proteins to nitrocellulose membranes. Also, methanol in the transfer buffer could restrict the elution of proteins from the gel. Decreasing the amount of methanol results in increased transfer efficiency, but it can also decrease the binding of proteins to the membrane. Hence, the right amount of SDS and methanol in the transfer buffer must be determined to get the optimal transfer conditions. The apparatus is cleaned by simply wiping everything down with 70% ethanol.
Overall, the Bio-Rad Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell is simple to assemble and set up and is a fast way to transfer proteins from a gel to a membrane for Western blot analysis. Also, because the apparatus has a large platform, one can transfer multiple gels at once. Once optimal transfer conditions have been determined, this device is a snap to use.
Hee Chul Lee
Graduate Student
Dept. of Biochemistry
NYU School of Medicine