Western Blot Transfer Methods

12 minute read


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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.

Wet transfer setup for electrophoresis
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, 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. 
  • Quick Tip: Transfer buffer from Azure Biosystems is specially is formulated to increase protein transfer and protein retention on the membrane for optimal sensitivity.

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.

Azure Aqua during Western blotting experiment
The Azure Aqua Transfer Cell is used for transferring two mini gels to membranes for Western blotting experiments (wet transfer). The transfer cassettes and electrode core are colored for directionality which makes transferring easy and straightforward. You can perform a quick 1-hour transfer or you can transfer overnight at a lower voltage.

"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).

Semi-dry transfer stack for electrophoresis
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.

  • Quick Tip: 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. 

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.

  • Quick Tip: Even 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.

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.

Want to try a new blocking buffer? We offer free samples!

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.

There’s an application note for that! Check out our app note “Wet or Dry?” for a variety of buffer recipes.

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