Efficient transfer of proteins out of a gel onto a membrane is critical when performing a Western blot. Inefficient transfer of a protein may skew results or cause the protein to become undetectable on the blot. High molecular weight proteins are known to be difficult to transfer out of the gel. Alternatively, low molecular weight proteins may transfer too readily, resulting in transfer of the protein through the membrane. Several parameters are critical in determining efficient transfer of proteins including transfer apparatus, voltage, heat and the transfer buffer.
This article provides a brief overview of the transfer process and a discussion of transfer buffers.
How transfer works
Prior to analysis of proteins by Western blotting, proteins must be eluted from the gel onto a solid support (the membrane) for detection by antibodies. Similar to the electrophoresis of proteins through the gel, proteins can be induced to migrate out of the gel using an electric field (termed electrophoretic transfer). In most transfer systems, negatively charged proteins move out of the gel towards the positively charged electrode.
Transfer can be done either under wet conditions in a tank apparatus or under semi-dry conditions on a solid support (see Types of Apparatuses). Regardless of the type of apparatus, the transfer is performed by surrounding the gel and membrane together with filter paper (often referred to as “the stack” or “the sandwich”) and placing them between two electrodes. Proteins will migrate out of the gel onto the membrane following the current applied through a transfer buffer. The protein size, shape and pH, gel percentage and ionic strength of the transfer buffer all affect the elution of protein out of the gel.
Types of transfer apparatuses
There are two main types of apparatuses used for transfer: wet transfer (also called tank) and semi-dry transfer. Both types of transfer procedures have advantages and disadvantages.
In wet transfer, the gel/membrane/filter paper stack is submerged completely in transfer buffer within a holding tank. A plastic cassette holds the stack in place and the cassette is placed between the electrodes and covered with transfer buffer. Electrodes can be either wire or plate.
Wet transfer works well for most routine protein work and is efficient at allowing transfer of proteins of all sizes (different transfer buffers can be used to optimize transfer of proteins – see Transfer Buffers). Wet transfer is highly recommended over semi-dry transfer when quantitative analyses are going to be performed downstream. With a proper cooling system, wet transfer can be performed for extended times (e.g. 24 h) at low voltage or rapidly (~1-2h) when using higher voltage.
Heating of the transfer buffer can be a major issue when using wet transfer systems. Local temperature increases can decrease the resistance of the transfer buffer resulting in inconsistent transfer across the gel. High heat can also result in breakdown of the gel itself. Most wet transfer systems are equipped with cooling mechanisms. These can be as simple as an ice block placed in the tank or as complex as a cooling coil attached to an external mechanism. Transfers are also often performed with pre-chilled transfer buffer in a cold room if space allows.
Another disadvantage to the wet transfer system is the volume of transfer buffer that is required to submerge the transfer stack. Transfer buffer should never be reused nor diluted; thus large quantities of buffer are required in active labs.
In semi-dry transfer, the filter paper is pre-soaked in transfer buffer, which provides the buffer required for transfer. The stack is placed directly between plate electrodes. Because the distance between the electrodes is minimal high electric field strengths are achieved and transfer is rapid (< 1h).
There are several disadvantages to semi-dry transfer that limit its utility. While semi-dry transfer works well for some proteins, the high intensity blotting conditions can cause smaller proteins to migrate through the membrane without becoming bound. Although transfer can be rapid, longer transfer times cannot be utilized, as the buffering capacity is low. Decreased transfer times can result in inefficient transfer of higher molecular weight proteins out of the gel. However, transfer of higher molecular weight proteins can be improved with the use of discontinuous buffer systems (see Types of Transfer Buffers).
Transfer buffers: An overview
Transfer buffers effectively elute proteins from the gel and promote binding of the proteins to the membrane. The choice of transfer buffer to be used is influenced by the type of gel, the blotting application, the membrane and the physical characteristics of the protein of interest.
Transfer buffer components
Transfer buffers generally contain relatively few components. However, buffer components should be optimized for the particular blotting system being used (e.g. wet transfer versus semi-dry), the type of gel used during electrophoresis, the type of membrane used and the protein of interest.
All transfer buffers must contain a strong buffering agent that maintains the conductivity and the pH during transfer. Typical buffers include Tris, CAPS or carbonate. Transfer buffers should never be diluted, as dilution will decrease the buffering capacity.
In native gels, the ability of the protein to transfer to the membrane is dependent on the pI of the protein relative to the pH of the buffer. If the pI is greater than the pH of the buffer, then the protein will migrate out of the gel towards the negative electrode. However, if the pI is less than the pH of the buffer, migration will be inhibited and the pH of the buffer will need to be adjusted to promote efficient transfer.
Alcohol (e.g. methanol) may be added to the transfer buffer to foster binding of proteins to the membrane by removing SDS from SDS-protein complexes. Note: alcohol can have a negative effect as it can cause protein precipitation or cause basic proteins to become positively charged or neutral, thus inhibiting transfer. The percent of alcohol used can be adjusted to aid in transfer of a protein.
Only high-quality, analytical grade alcohol should be used in transfer buffers as impurities in alcohols can result in poor transfer. In many instances ethanol can be substituted for methanol without affecting transfer efficiency, although this should be determined empirically.
Alcohol is not used when transferring proteins from native gels or when transferring acidic or neutral proteins.
SDS (sodium dodecyl sulfate or sodium laurel sulfate)
SDS detergent may also be included in transfer buffers to promote elution of proteins out of the gel. However, SDS inhibits binding of proteins to the membrane, particularly when using nitrocellulose membranes. Therefore PVDF membranes should be used when SDS is added to the buffer.
Note: Alcohol and SDS have opposing effects on proteins during transfer. Alcohol promotes binding of proteins to the membrane, but can inhibit elution of the protein out of the gel. In contrast, SDS promotes elution of proteins, but can inhibit binding to the membrane. Both of these components can be adjusted to increase the efficiency of transfer.
Types of Transfer Buffers
Towbin and Bjerrum Schafer-Nielsen Buffers
Towbin developed the most commonly used transfer buffer in 1979. The standard buffer consists of 25 mM Tris, 192 mM glycine, pH 8.3 and 20% methanol. Towbin buffer is a general, all-purpose buffer that is compatible with SDS-PAGE, Tris-Tricine and two-dimensional gels. Both nitrocellulose and PVDF membranes may be used with Towbin buffer. The standard buffer efficiently transfers low molecular weight proteins and high molecular weights may also be transferred readily when the buffer is supplemented with SDS (0.025-0.1%, depending on the protein).
The Bjerrum Schafer-Nielsen buffer was developed as a Towbin-like buffer to enhance transfer when using semi-dry blotting apparatuses. This buffer contains 48 mM Tris, 39 mM glycine, pH 9.2 and 20% methanol.
CAPS (3-[cyclohexylamino]-1 propane sulfonic acid) Buffer
CAPS buffer is often used when blotting basic proteins or for blotting of proteins prior to N-terminal sequencing, although CAPS is compatible with the same applications and protein characteristics as Towbin buffer (SDS-PAGE, Tris-Tricine and two-dimensional gels; nitrocellulose and PVDF membranes; wet or semi-dry transfer). The buffer composition is 10 mM CAPS, pH 11 and 10% methanol.
Dunn Carbonate Buffer
The Dunn buffer uses carbonate for higher efficiency transfer of basic proteins. It can also enhance the ability of some antibodies to recognize and bind to antigenic sites on proteins. Dunn buffer is compatible with SDS-PAGE and two-dimensional gels. The buffer is composed of 10 mM NaHCO3, 3mM Na2CO3, pH 9.9 and 20% methanol.
Discontinuous Tris-CAPS Buffer System (for Semi-Dry Transfer)
The discontinuous Tris-CAPS buffer system takes advantage of a unique property of semi-dry blotting: the capability of using two different buffers during transfer (called discontinuous buffer system). The filter papers placed on either side of the membrane are soaked in two different buffers to facilitate transfer: the filter paper assembled on the membrane side (anode) of the blot contains methanol while the filter paper on the gel side (cathode) of the blot contains SDS. This allow SDS to promote migration of proteins out of the gel without inhibiting their ability to bind to the membrane while also allowing the alcohol to foster protein binding to the membrane without inhibiting migration of the proteins out of the gel. The stock buffer is composed of 60 mM Tris and 40 mM CAPS, pH 9.6 and is supplemented with either 15% methanol or 0.1% SDS.
Acetic acid (0.7%) may be used as a transfer buffer when transferring proteins from gels for isoelectric focusing, transferring a native-PAGE gel or when working with basic proteins.
Note: the use of acetic acid will cause the proteins to become positively charged and migrate towards the cathode side of the gel.
FLASHBlot Transfer Buffer
Several companies also market non-traditional transfer buffers. For example, Advansta recently released the FLASHBlot Transfer Buffer. Designed for wet transfer systems, FLASHBlot Transfer Buffer is useful for transferring and retaining both high and low molecular weight proteins on the membrane with equal efficiency. Additionally, problems arising from heating of the transfer buffer are nonexistent as transfers are performed in 15 minutes when using FLASHBlot Transfer Buffer.