When to use Wet, Semi-Dry and Dry Transfers for Western Blots

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Transfers Western Blotting

Choosing the right transfer method can determine your Western blot’s success. The first step to Western blotting is separating the proteins in a sample by size, using a denaturing process called gel electrophoresis.

How does Western blot transfer work?

In Western blotting, after the electrophoresis step, is the transfer step. The transfer step is important to Western blotting because it moves the separated proteins from the gel onto a solid support matrix using a membrane (either nitrocellulose or polyvinylidene difluoride (PVDF)). This is where the blot will form.

Cases where you would not need to transfer the gel

Sometimes, protein separation is not required, so your sample may be directly added to the membrane using an approach called “dot blotting.”

Three types of Western blot transfers

Your choice of transfer comes 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 (Table 1). So, wet or dry – which is the best transfer method for your Western blot? Let’s go over when to use each transfer method to best prepare you for your next Western blot procedure.

Table of contents

Table 1. When to know when to choose which Western blot transfer method

ScenerioWhich transfer method to use
You want to gain quantitative information from your Western blotWet transfer
Wet transfers allow you to customize the time, temperature, voltage, or buffer to best suit your protein of interest.
Saving time and reagents is equally importantSemi-dry transfer
In a semi-dry transfer, the only buffer used is the one that saturates the stack components (Figure 2).
You're short on timeDry transfer
Dry transfers typically only take around ten minutes to finish. They are also the least quantitative Western blot method.

How wet transfers for Western blotting work

If you want to gain quantitative information from your Western blot, you should do a wet transfer using a tank. Wet transfers are performed in a tank filled with transfer buffer (Figure 1). Most transfer systems, like the Azure Aqua Transfer Tank, have room for two precast or handcast gels. Tanks are routinely used in wet labs for Western blot transfers.

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.

The transfer buffer used for wet transfer protocols is traditionally a Tris-glycine buffer, which contains methanol, but you may also use other buffers too. In certain cases, SDS may be added to the buffer to aid the transfer of large proteins. 

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

Pros:

  • 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 also be reduced to slow the transfer process for an overnight run, or increased to complete the transfer in an hour or two.
  • Overall less expensive than other transfer methods
  • Can use multiple buffers to optimize transfers
  • Transfers broad molecular weight range at one time
  • Extended transfer is possible
  • Can be used for quantitative Westerns

Cons:

  • 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 or cooling units in the tank.
  • Another disadvantage to wet transfers is they require a large volume of transfer buffer. If you regularly conduct a large number of transfers, reagent consumption can become an issue. Because of the larger volume of usage, you will also accumulate more hazardous waste. To conserve reagents, semi-dry transfer methods can be used.
  • Wet transfer can also take up to one hour. With some systems, you can also run transfers overnight at a low voltage.
  • Cooling mechanism and/or cold room space is required during transfer.

How semi-dry Western blot transfer methods work

In a semi-dry transfer, the only buffer used is the one that saturates the stack components. The membranes and gels will be placed between wet filter papers (a sandwich) that will be in direct contact with the electrodes (Figure 2). Semi-dry transfers should be your choice if saving time and reagents is your first priority.

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. This stack is placed directly between two electrode plates.
  • 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.

Pros:

  • Overall easy to setup. Good for performing large numbers of blots where you are analyzing the same protein.
  • With semi-dry transfers, transfer times are reduced to about an hour, but may be as short as five  minutes with rapid semi-dry transfer protocols.
  • The hazardous waste from transfer buffer is minimized because only small volumes are used each time.

Cons:

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 entirely through the membrane.

  • To compensate, you can use discontinuous buffer systems. With these systems, the two pieces of filter paper on either side of the stack are equilibrated in different buffers. 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.
  • High intensity field strength may cause low molecular weight proteins to migrate through membrane.
  • Difficulty in transferring high (>120 kDa) molecular weight proteins
  • Not recommended for quantitative Western blots
  • 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.

How “dry” Western blot transfer methods work

With dry transfer, the gel is placed between the preassembled stacks, that already contain the transfer membrane and proprietary buffer matrices you’ll need. Because of this convenience, if you are short on time, dry transfers are the best option.

Pros:

  • Dry Western blot transfer systems do not use transfer buffer at all, which saves time.
  • Can be completed in as little as ten minutes when using newer models.

Cons:

  • There is no opportunity to customize or optimize your solutions based on your protein of interest.
  • With dry transfers, preassembled stacks must be purchased ahead of time. This adds to reagent and consumable costs.

Remember: 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, the transfer method you choose will depend on several factors of your Western blotting 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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