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We investigated the feasibility of repeated use of transfer buffer containing methanol in electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to polyvinylidene difluoride (PVDF) membrane using a prestained protein marker of broad molecular sizes. Transfer of the antitumor protein p53 in HEK293T cell extracts, using fresh and used transfer buffer, followed by detection with anti-p53 antibody was also performed to test detectability in immunoblot. Results from these experiments indicate that the transfer buffer can be reused at least five times and maintain a similar extent of protein transfer to PVDF membrane. Repeated use of the transfer buffer containing methanol will significantly reduce the volume of hazardous waste generated and its disposal cost as well as its adverse effect on environment.
Protein transfer from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels to membrane is a useful technique for detection, quantification, and characterization of a specific protein in cell/tissue extracts as well as in purified samples.1–4 It is also used in proteomics and epigenetics studies for protein expression and modifications and/or protein–protein interactions, indicating its wider applications.4 Of the three different methods developed for protein transfer, namely simple diffusion,5 vacuum-assisted solvent flow,6 and electrophoretic elution,7 the latter (popularly known as Western blot) is widely used and is the method of choice in most laboratories.8 In many laboratories, researchers perform Western blot on a regular basis and often carry out this procedure several times a week or even a day. Western blot can be carried out according to semidry or wet transfer procedure. A number of buffers are recommended in the Bio-Rad protocols for wet electrophoretic transfer, such as Tris-glycine buffer of varying concentrations, pH (8.3–9.2) and SDS (0.025%–0.1%) with or without 20% methanol or bicarbonate buffer (pH 9.9), SDS (0.025%–0.1%) with or without 20% methanol, 1X TBE (90 mM Tris-borate, 1 mM EDTA) and 1X TAE (40 mM Tris-acetate, 1 mM EDTA). The Tris-glycine buffer, pH 8.3, containing 0.1% SDS with 20% methanol is widely used in laboratories, primarily because the buffer without methanol is the universal buffer for SDS-PAGE gel running. The presence of methanol in the transfer buffer serves two main purposes: It promotes dissociation of SDS from the protein and dramatically improves adsorption of proteins onto membranes in the presence of SDS, although these effects may vary with proteins.9 Of the two membrane types used in most laboratories, namely nitrocellulose and PVDF, the latter is preferred for its better protein-binding capacity.
When wet electrotransfer is performed several times, usually requiring 0.8–1.0 L transfer buffer per run, a large volume of waste transfer buffer accumulates. While methanol promotes important benefits, as noted above, its presence in the transfer buffer renders it toxic and, since the hazardous waste produced cannot be flushed down the drain, considerable resources are needed to assure its safe disposal. In a recent study, reduced (10%) methanol was used to lessen its presence in the transfer buffer.9 Villan-ueva10 showed that the rubbing alcohol 2-propanol could be successfully substituted for methanol in transfer buffer for wet electrotransfer of proteins from SDS-PAGE gels to membranes. However, as with all organic molecules, a large volume of 2-propanol in the transfer buffer becomes a hazardous waste.
In this study, we show that transfer buffer containing methanol can be successfully reused multiple times in wet electrotransfer/Western blotting for protein transfer from SDS-PAGE gels to PVDF (Sequiblot, Bio-Rad, Hercules, CA) membranes. Recycling transfer buffer in this manner significantly reduces the volume of methanol containing hazardous, toxic waste generated in each laboratory, thus reducing the cost of waste disposal as well as its adverse effects on the environment.
In order to investigate whether the transfer buffer can be used repeatedly in protein electrotransfer or Western blotting, BenchMark Prestained Protein Ladder (Invitrogen, Carlsbad, CA) (6 μL/lane) was run on 10% SDS-PAGE gels at a constant 150 V until the bromophenol blue dye was close to the bottom end. After completion of the running, the gel was gently shaken in the transfer buffer (fresh or used) for 30 min, then assembled into gel transfer cassette. PVDF membrane cut to the gel size was soaked in methanol, then rinsed with distilled water and transfer buffer. After assembly, gel sandwich was placed in transfer apparatus along with ice pack and transferred at 100 V for 1 h using wet electrotransfer procedures according to the manufacturer’s (Bio-Rad) instruction. Following each transfer, approximately 600–650 mL buffer was saved at 6°C until further use. The transfer was carried out using transfer buffer that had been prepared fresh (1x) up to seventh use (7x), each time approximately 150–200 mL of fresh transfer buffer was added to make up to the total minimum volume of 800 mL. All other aspects of the transfer were held constant from run to run. It should be noted here that unlike the fresh transfer buffer, the used transfer buffer forms a precipitate during storage at 6°C which dissolves when brought to room temperature by warming in a 37°C water bath.
The success of the used transfer buffer in protein transfer and/or binding was evaluated by comparing intensities of the protein bands present in the Benchmark prestain marker (Invitrogen, Carlsbad, CA). As shown in Figure 1A, no difference in intensity was observed in all protein bands present in the marker until the fifth use of the transfer buffer, after which a slight decrease in protein transfer was apparent. Figure 1B shows transfer and detection of the antitumor protein p53 in HEK293T cell extracts using rabbit anti-p53 antibody (Sigma) in immunoblot, where a slightly less p53 band intensity, but still a quite strong signal, was observed in the 5X transfer buffer compared with the fresh transfer buffer (1X). These results indicate that the transfer buffer containing 20% methanol can be reused multiple times without significant loss in protein transfer as well as detection in immunoblot using antibodies. We tried reusing transfer buffer containing 10% methanol up to three times and although we used the same amount (6 μL) of the Benchmark marker, the transfer (or binding) was not as efficient as with 20% methanol even with the fresh buffer (1X).
In this study, we were able to successfully transfer a prestained protein marker from SDS-PAGE gels to PVDF membranes with as many as seven times reused transfer buffer. This reuse of the methanol containing transfer buffer will significantly reduce the volume of hazardous, methanol containing waste generated and its subsequent disposal cost.