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Choosing a Transfer Method for Your Western Blot

Wet or dry – which is best transfer method for your Western blot? Choosing the right one can determine whether your Western blot is a success. Western blot transfers can be “wet”, “semi-dry”, or “dry” – which should you use and why? The choice may come down to whether you need quantitative information from your blot, whether time and cost are considerations, and whether your protein is finicky and requires you to customize transfer conditions.

When should you use “wet” Western blot transfers?

“Wet” transfers are performed in a tank filled with transfer buffer (described in Figure 1).

Figure 1. Wet transfer setup. A “stack” is built in which the gel is placed next to a membrane (nitrocellulose or PVDF), both of which have been pre-equilibrated in transfer buffer. Blotting papers and sponge, which have also been pre-soaked in transfer buffer, are added to the outside of the stack so the stack can be held firmly within a cassette that is suspended in the transfer buffer–filled tank. Electrodes on the cassette allow an electric current to be run through the stack so the proteins migrate from the gel to the membrane.

If you want to gain quantitative information from your Western blot, in many cases you should do a wet transfer. Wet transfers are highly customizable – the time, temperature, voltage, and buffer can be varied to suit the protein of interest and to achieve complete transfer of a broad range of proteins. For example, longer transfer times may be used to allow larger proteins to fully migrate out of the gel, while shorter transfer times can prevent loss of low-molecular weight proteins that may otherwise migrate through the membrane entirely. The voltage can be reduced to slow the transfer process for an overnight run, or increased to complete the transfer in an hour or two.

Transfer buffer for wet transfer protocols is traditionally a Tris-glycine buffer containing methanol. Other buffer systems may be tried, and SDS may be added to the buffer to aid the transfer of large proteins. Azure Biosystems’ Transfer Buffer is formulated to increase protein transfer and protein retention on the membrane for optimal sensitivity. See the Application Note “Wet or Dry?” for a variety of buffer recipes.

One disadvantage of wet transfers is that heat is generated during the transfer. This can contribute to inconsistent transfers and to breakdown of the gel. To combat the effects of excess heat, wet transfers are often conducted in a cold room and/or with ice packs in the tank.

Another disadvantage to wet transfers is that they require a large volume of transfer buffer. For labs conducting a large number of transfers, reagent consumption can become an issue. To conserve reagents, semi-dry transfer methods can be used.

When should you use “semi-dry” Western blot transfer methods?

“Semi-dry” transfers should be your choice if saving time and reagents is your first priority. In a semi-dry transfer, the only buffer used is that which saturates the stack components (Figure 2).

Figure 2. Semi-dry transfer setup. In a semi-dry transfer, the stack consists of gel and membrane placed between two pieces of filter paper, all equilibrated in transfer buffer, and this stack is placed directly between two electrode plates.

With semi-dry transfers, transfer times are reduced to about an hour, but may be as short as 5 minutes with rapid semi-dry transfer protocols. However, transfer times cannot be extended for proteins that do not transfer completely with the standard protocol. The stack can dry out and the buffer capacity of the small amount of transfer buffer will be exhausted if transfer times are too long.

Semi-dry transfers run into difficulties at extreme ends of the protein size range. Large proteins may not transfer out of the gel quantitatively in the short transfer time available while small proteins may transfer through the membrane entirely. To compensate, discontinuous buffer systems, in which the two pieces of filter paper on either side of the stack are equilibrated in different buffers, can be used. For example, buffers can be chosen to help transfer difficult proteins out of the gel, and/or to improve retention of proteins in the membrane. However, though discontinuous buffer systems can help improve transfer across a larger range of protein sizes, semi dry transfers are not recommended for quantitative Western blotting.

What is “dry” Western blot transfer?

“Dry” Western blot transfer systems do not use transfer buffer at all. Instead, the gel is placed between purchased, preassembled stacks containing the transfer membrane and proprietary buffer matrices. Dry transfers can be completed in less than 10 minutes. Because preassembled stacks must be purchased, there is no opportunity for customization or optimization based on the protein of interest.

In summary, which transfer method you choose will depend on your experiment and how you balance the importance of speed and/or reducing reagent consumption vs needing quantitative transfers or to customize the protocol for a difficult protein. Wet Western blot transfers are highly customizable and are recommended for quantitative Western blotting but consume a lot of reagents. Semi-dry Western blot transfers conserve time and reagents, but may not allow quantitative transfer for all proteins, especially those that are very small or very large. Dry Western blot transfers are the fastest of all and require no buffer preparation, but do not allow much room for optimization. For more information about transfer methods and their advantages and disadvantages, including transfer buffer recipes, check out this Application Note from Azure.

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