The first step to Western blotting is separating the proteins in a sample by size using denaturing gel electrophoresis. After electrophoresis, the separated proteins are transferred onto a solid support matrix using a membrane (nitrocellulose or polyvinylidene difluoride (PVDF)). Sometimes, protein separation is not required, so your sample may be directly added to the membrane using an approach we call dot blotting.
So, wet or dry – which is the best transfer method for your Western blot? Choosing the right one can determine your blot’s 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, time and cost, or if your protein is finicky and requires you to customize transfer conditions. Let’s go over when to use each transfer method to best prepare you for the next Western blot procedure you run.
When to use “wet” (or Tank) transfers for Western blotting
“Wet” transfers are performed in a tank filled with transfer buffer (described in Figure 1). Most transfer systems, like the Azure Aqua Transfer Tank, have room for two precast or handcast gels.
If you want to gain quantitative information from your Western blot, 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.
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 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.
"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 the one that saturates the stack components (Figure 2).
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.
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.
When to use “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.
Western blot transfers are customizable!
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.
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In summary, which transfer method you choose will depend on several factors of your experiment. One of the factors is 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. Check out the resources below if you’re still having trouble, or send us a message using the form on this page. Cheers for now!
Frequently Asked Questions about western blot transfers
The reason for blocking the membrane before and during incubation of the primary antibody is due to the membranes being “sticky” to any protein. We “block” in order to cover up all parts of the membrane that don’t have protein on them, including any region in between lanes, etc. When we add the antibodies, they will only bind to the protein of interest, not the blank membrane. Blocking ensures we don’t get signal from primary antibodies sticking to the membrane. We will only get signal from primary antibodies binding to their protein of interest.
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The blocking agent may be protein or non-protein based. Some of the more commonly used blocking agents include: normal serum, Bovine serum albumin, non-fat, dry milk, Polyvinylpyrrolidone, or Tween 20. Read more about blocking agents in this application note.