Western Blot Protein Transfer

Comparison of wet, semi-dry and dry transfer methods

Efficient and reliable protein transfer from the gel to the blotting membrane is the cornerstone of a successful western detection experiment. Accuracy of results is dependent on the transfer efficiency of the western blotting method. Traditional wet transfer offers high efficiency, but at a cost of time and effort. Semi-dry blotting provides convenience and time savings, with flexibility to use multiple types of buffer systems or pre-assembled stacks that are buffer free. However, semi-dry transfer can have a lower efficiency of transfer of large molecular weight proteins (>300 kDa). Dry electroblotting offers both high quality transfer combined with speed as well as convenience since added buffers are not required for dry electroblotting.

Three major ways to transfer proteins from SDS-PAGE or native gels to nitrocellulose, PVDF or nylon membranes:

• Wet tank electrotransfer
• Semi-dry electrotransfer
• Dry electrotransfer

When performing a wet transfer, the gel is first equilibrated in transfer buffer. The gel is then placed in the “transfer sandwich” (filter paper-gel-membrane-filter paper), cushioned by pads and pressed together by a support grid. The supported gel sandwich is placed vertically in a tank between stainless steel/platinum wire electrodes and filled with transfer buffer.

Multiple gels may be electrotransferred in the standard field option, which is performed either at constant current (0.1 to 1 A) or voltage (5 to 30 V) from as little as 1 hour to overnight. Transfers are typically performed with an ice pack and at 4°C to mitigate the heat produced. A high field option exists for a single gel, which may bring transfer time down to as little as 30 minutes, but it requires the use of high voltage (up to 200 V) or high current (up to 1.6 A) and a cooling system to dissipate the tremendous heat produced.

Transfer efficiencies of 80–100% are achievable for proteins between 14–116 kDa. The transfer efficiency improves with increased transfer time. With increasing time, however, there is a risk of over-transfer (blew through) of the proteins through the membrane, especially for lower molecular weight (<30 kDa) proteins when using membranes with a larger pore size (0.45 µm).

For semi-dry protein transfer, the transfer sandwich is placed horizontally between two plate electrodes in a semi-dry transfer apparatus. For this semi-dry transfer, it is very important that the gel is pre-equilibrated in transfer buffer. To maximize the current passing through the gel instead of around the gel, the amount of buffer available during transfer is limited to that contained in the sandwich, so it is helpful if the extra-thick filter paper (~3 mm thickness) and membrane are also sufficiently soaked in buffer. Likewise, it is key that the filter paper sheets and membrane are cut to the size of the gel.

One to four gels may be rapidly electroblotted to membranes. Methanol may be included in the transfer buffer, but other organic solvents, including aromatic hydrocarbons, chlorinated hydrocarbons and acetone, should not be used to avoid damage to the semi-dry blotter. Electrotransfer is performed either at constant current (0.1 up to ~0.4 A) or voltage (10 to 25 V) for 10 to 60 minutes. Methanol-free transfer buffers are recommended to reduce transfer time to 7 to 10 minutes. Transfer efficiencies of 60 to 80% may be achievable for proteins between 14 and 116 kDa, longer transfer times are required to transfer higher molecular weight proteins.

Dry electroblotting methods use a specialized transfer sandwich containing innovative components that eliminate use of traditional transfer buffers. A unique gel matrix (transfer stack) that incorporates buffer is used instead of buffer tanks or soaked filter papers. The high ionic density in the gel matrix enables rapid protein transfer. During blotting, the copper anode does not generate oxygen gas as a result of water electrolysis, reducing blot distortion. Conventional protein transfer techniques, including wet and semi-dry, use inert electrodes that generate oxygen. Typically, transfer time is reduced by the shortened distance between electrodes, high field strength and high current.