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The process of transferring proteins from a gel to a membrane while maintaining their relative position and resolution is known as blotting. Blotting can be achieved in three different ways:
Simple diffusion (Kurien and Scofield, 1997) is accomplished by laying a membrane on top of the gel with a stack of dry filter paper on top of the membrane, and placing a weight on top of the filter paper to facilitate the diffusion process (Kurien and Scofield, 2003). This method can be used to transfer proteins from one gel to multiple membranes (Kurien and Scofield, 1997), obtaining several imprints of the same gel. The major disadvantage of the diffusion method is that it only transfers 25-50% of the proteins as compared to electroblotting (Chen and Chang, 2001).
Vacuum-assisted solvent flow (Peferoen et al.,1982) uses the suction power of a pump to draw separated proteins from the gel onto the membrane. Both high and low molecular weight proteins can be transferred by this method; however, a smaller pore size membrane (0.2 :m) may be needed for proteins with MW < 20 kDa, since they are less readily adsorbed by the 0.45 μm membrane (Kurien, 2003). Vacuum blotting of proteins out of polyacrylamide gels is uncommon and is mostly used for nucleic acid transfer from agarose gels.
Electrophoretic elution, or electrotransfer (Towbin et al., 1979) is by far the most commonly used transfer method. The principal advantages are the speed and completeness of transfer compared to diffusion or vacuum blotting (Kurien et al, 2003).
The two commonly used electrotransfer techniques are tank transfer and semi-dry transfer. Both are based on the same principles and differ only in the mechanical devices used to hold the gel/membrane stack and applications of the electrical field.
Tank transfer (see the "Tank (wet) Transfer System" figure below), is the traditional technique where the gel/membrane stack is completely immersed in a buffer reservoir and then current is applied. It is an effective but slow technique, using large volumes of buffer. Tank systems are typically run at constant voltage; mixing of the buffer during transfer keeps the current relatively constant.
Semi-dry transfer (see the figure below) replaces the buffer reservoir with layers of filter paper soaked in buffer. Because the plate electrodes are in direct contact with the filter papers, the field strength across the gel is maximized for fast, efficient transfers. This technique is as effective and far quicker (15-45 minutes) than tank transfer. Most semi-dry transfer methods use more than one buffer system to achieve efficient transfer of both large and small proteins. However, semi-dry blotting systems have lower buffering capacity and thus are inappropriate for prolonged transfers. Semi-dry transfer is the preferred method for blotting large 2-D gels. Semi-dry blotters are typically run at constant current; the voltage normally increases during the transfer period.
For semi-dry transfer systems, it is important that the filter papers and membrane are cut to the same size as the gel so that the current is forced to flow through the gel. Otherwise, the current will short-circuit through overlapping filter paper around the edges of the gel. In both types of transfer systems, extra caution should be taken to prevent introduction of air bubbles anywhere between the filter paper, gel and membrane. Bubbles prevent transfer and cause "bald spots" (i.e., areas of non-transfer) on the blot.
Anode buffer I neutralizes excess protons generated on the surface of the anode plate. Anode buffer II contains Tris at the same pH as anode buffer I, but at a reduced concentration of 25 mM. The cathode buffer contains e-aminocaproic acid, which serves as the trailing ion during transfer and is depleted from the cathode buffer as it migrates through the gel toward the anode. Review manufacturer’s recommendations for single buffers in a semi-dry system.
Although the buffer systems defined above are suitable for the majority of protein transfers, the literature contains many variations suited to different applications. One of the most significant variations was the recommendation of 10 mM CAPS buffer at pH 11 for protein sequencing applications (Matsudaira, 1987). The glycine used in Towbin buffer and carried over from the gel running buffer caused high backgrounds in automated protein sequencers employing Edman chemistry. By changing the transfer buffer composition, this artifact was significantly reduced. Any modification to the buffer strength and composition should be made with care to ensure that the transfer unit does not experience excessive heating.
The methanol added to transfer buffers has two major functions:
Polyacrylamide is a hydrogel that has the capacity to absorb water. In pure water, the gel’s size increases in all dimensions by a considerable amount. The degree of swelling also depends on the concentration of acrylamide used in the gel. High concentration gels expand more than low concentration gels. Gradient gels highlight this effect quite dramatically with the more concentrated zone at the bottom expanding much more than the top. A gel that starts out rectangular may become trapezoidal. The methanol added to the transfer buffer minimizes gel swelling, and transfer protocols normally include an equilibration step to achieve dimensional stability. At concentrations of 10% to 20%, dimensional stability can be achieved fairly rapidly. At lower methanol concentrations, more time is required for equilibrium to be achieved. If dimensional changes occur during transfer, the resolution of the proteins may be lost. For high MW proteins with limited solubility in methanol, elimination of the methanol can result in a significant increase in protein transfer efficiency, but this may necessitate a longer equilibration time to ensure dimensional stability.
The second function of methanol is critical for transfer of proteins from gels containing SDS. Methanol helps to strip complexed SDS from the protein molecules (Mozdzanowski and Speicher, 1992). Although the SDS is necessary for resolution of individual proteins on the gel, it can be extremely detrimental to effective blotting. First, by imparting a high negative charge density to a protein molecule, the SDS causes the protein molecule to move very rapidly through the membrane, reducing the residence within the pore structure and minimizing the opportunity for molecular interaction. Second, by coating the protein molecule, the SDS limits the ability of the protein to make molecular contact with the PVDF. These effects increase as the MW of the protein decreases. Methanol reduces both effects by stripping off the SDS and increasing the probability that a protein molecule will bind to the membrane.
When the transfer of BSA was monitored over two hours in a standard tank transfer system, the data suggested that within a single protein band there is more than one population of molecules transferring from the gel (see the figure “Electrotransfer of BSA,” below ). About 90% of the BSA is eluted from the gel during the first 60 minutes, with an additional 7% eluting in the last 60 minutes. During the first 15 minutes, part of the eluted BSA adsorbed to the Immobilon-P transfer membrane while the remainder passed through and adsorbed to the Immobilon-PSQ transfer membrane. BSA that eluted after 15 minutes adsorbed almost exclusively to the sheet of Immobilon-P transfer membrane. The simplest interpretation is that the BSA bound to a high level of residual SDS eluted from the gel rapidly and was unable to adsorb to Immobilon-P transfer membrane. BSA that eluted more slowly was able to adsorb to the Immobilon-P transfer membrane.
Although removal of SDS from a gel is generally the best approach for routine blotting, there are instances where addition of low amounts of SDS to the transfer buffer is worth considering when the proteins to be transferred have low solubility in the absence of SDS. Proteins from cellular membranes may be very hydrophobic and can precipitate within the polyacrylamide as the SDS is removed. High MW proteins also may exhibit solubility problems in the absence of SDS, especially after being exposed to the denaturing conditions of the gel sample buffer and the methanol used in the transfer buffer. Supplementation of the transfer buffer with SDS can be used to maintain sufficient solubility to permit elution from the gel (e.g., Towbin and Gordon, 1984, Otter et al., 1987; Bolt and Mahoney, 1997). The SDS concentration in the transfer buffer should not exceed 0.05%, and sufficient equilibration time should be allowed to remove all excess SDS from the gel.
Other methods employed to improve the transfer efficiency of high molecular weight proteins were prolonged blotting time, up to 21 hours (Erickson et al., 1982), or the use of a composite agarose- polyacrylamide gel containing SDS and urea (Elkon et al., 1984).
The appropriate current and transfer time are critical for successful blotting. Insufficient current and/or time will result in incomplete transfer. Conversely, if the current is too high, the protein molecules may migrate through the membrane too fast to be adsorbed. This can be a significant problem for smaller proteins. Usually, blotting systems come with manufacturer’s recommendations for current and transfer time that should be used as a guideline. Optimization may still be required depending on the gel percentage, the buffer composition and the MW of the protein of interest. Generally, long transfer times are best suited for tank systems, which normally require cooling of the unit and internal recirculation of the transfer buffer. In semi-dry transfer, however, prolonged blotting may result in buffer depletion, overheating and gel drying. If too much drying occurs, the unit can be damaged by electrical arcing between the electrode plates.
The pH of the transfer buffer is another important factor. If a protein has an isoelectric point equal to the buffer pH, transfer will not be promoted. To alleviate this problem, the higher pH buffers such as CAPS or the lower pH buffer such as acetic acid solutions can be used.
In the early days of protein blotting (late 1970s, early 1980s), most protocols called for equilibration of the gel for 30 minutes prior to blotting. Standard gel sizes of 5 inches or more on a side and minimum thicknesses >1 mm required extended equilibration to stabilize the size of the gel. As mini-gels became more common, equilibration times were shortened because there was less volume into which the water and methanol had to equilibrate.
Dimensional equilibrium can be reached in standard mini-gels within 5 to 10 minutes, but the kinetics of SDS stripping are significantly slower, so a minimum equilibration time of 15 minutes is recommended for most mini-gels. Note: For samples containing small peptides, the rapid migration of peptides can occur without electrical force. In this instance, equilibration of the gel in transfer buffer should be limited to less than 10 minutes.
In SDS-PAGE systems, the running buffer is supplemented with SDS. This SDS concentrates from the cathode reservoir and runs into the gel behind the Bromophenol blue tracking dye. Since most gels are run until the tracking dye is at the bottom of the gel, all of the excess SDS remains in the gel and is carried over into the blotting procedure. If it isn’t allowed to diffuse out of the gel prior to transfer, it will interfere with protein adsorption. Equilibration times can be extended up to 30 minutes, and sufficient buffer should be used to reduce the SDS to a minimal level.
The effect of equilibration time on electrotransfer of BSA is shown in the figure on this page. In this study, radioactive BSA was resolved by SDS-PAGE, and the gels were equilibrated in transfer buffer for periods ranging from 0 to 30 minutes. Protein was transferred to Immobilon-P transfer membrane backed up with a piece of Immobilon-PSQ transfer membrane to adsorb any BSA not retained by the Immobilon-P transfer membrane. At the end of the transfer period, the BSA in the gel, on the primary blot (Immobilon-P transfer membrane) and on the back-up blot (Immobilon-PSQ transfer membrane) was quantified. Retention improved to 90% when the duration of the equilibration period was increased to 30 minutes. Other proteins have been found to behave similarly.
Although the previous section suggests that the selection of buffers and transfer conditions can be very complex, the tank transfer system defined by Towbin et al. (1979) and the semi-dry transfer system defined by Kyhse-Anderson (1984) work well for most protein samples. Both represent excellent starting points. If they prove less than optimal for a particular protein, though, transfer conditions can be tailored to fit the biochemical peculiarities of the protein. An interesting optimization strategy for the efficient transfer of proteins over a MW range from 8,000 to >400,000 kDa was demonstrated by Otter et al. (1987). The transfer buffer was supplemented with 0.01% SDS to maintain the solubility of high MW proteins and 20% methanol to enhance adsorption. The electrical field was applied in two phases. The first hour of transfer was at a low current density to slow the migration rate of low MW proteins and increase their residence time in the membrane. This was followed by a prolonged period at high current density to elute the high MW proteins.
When developing a new transfer protocol or working with a new sample type, the gel should be stained to verify that all of the proteins have completely eluted from the gel. It is also highly recommended to have a lane with pre-stained markers in each gel to monitor the transfer efficiency. Some proteins have limited solubility in typical transfer buffers, requiring modification of the buffer chemistry to prevent precipitation. Other proteins, such as histones and ribosomal proteins, are positively charged in standard transfer buffers and will migrate toward the cathode. These proteins can be successfully transferred by placing a sheet of Immobilon-P membrane on the cathode side of the gel. Staining the membrane after the transfer can also be helpful to ensure that the target protein is on the blot. See “Protein Visualization,” below, for information on stains compatible with immuno detection.
Another method to monitor protein transfer is to stain SDS-PAGE gels prior to electroblotting (Thompson and Larson, 1992). In this method, the gels are stained with either Coomassie Brilliant Blue after electrophoresis, or during electrophoresis using the Chroma-Glo™ visualization system (Promega). The transferred proteins remain stained during immunodetection, providing a set of background markers for protein location and size determination (Thompson and Larson, 1992).