Thermal Denaturation of Proteins for SDS-PAGE Analysis by Microwave Irradiation

One of the most useful tools available to researchers is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (3). It is a rapid and relatively simple method for resolving proteins and is routinely used to monitor proteins during purification, assess expression levels, estimate molecular weights and characterize multimeric proteins. A number of variations exist, but the method developed by Laemmli (5) is the most widely used. Sample preparation for SDS-PAGE analysis consists of denaturing the sample with heat in the presence of SDS and a reducing agent such as 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). This is achieved by mixing the sample with an appropriate volume of concentrated sample buffer (10). The heating step, usually 3–5 min at 100°C, can be performed by incubating the sample in a microcentrifuge tube in either boiling water or a heating block set at 100°C. This treatment destroys secondary and tertiary structure, solubilizes the protein, dissociates polypeptides and reduces disulfide bonds. There are safety drawbacks in both of these heating methods. When microcentrifuge tubes are heated to 100°C, the entire contents become heated, often resulting in the caps popping open as a result of pressure building up from the heated air within the tube. This can be particularly hazardous if the contents of the tube are radiolabeled, causing aerosols or even droplets of radioactive material to be spread over the surrounding area. Methods for overcoming the build-up of pressure include puncturing the cap of the tube with a needle, introducing another potential hazard, or the use of more expensive screw-cap tubes. In addition, conventional methods of heating result in the whole tube becoming hot, making handling difficult. We describe here a microwavebased heat denaturation method. The use of microwaves in laboratories has become commonplace, and they are regularly used to boil agarose, to melt agar and to warm serum and media before use in cell culture. Other applications include the rapid thawing of blood components (8), protein assays (1), fixing of samples for electron microscopy and immunohistochemistry (6), in situ end-labeling of DNA (7) and preparing plasmid (4,11) and genomic (2,9) DNA. To demonstrate the efficiency of microwave irradiation in the preparation of protein samples for SDS-PAGE analysis, we have used purified bovine γ-globulins (Sigma Chemical, Poole, England, UK) for the comparison of the microwave and heating-block methods of protein denaturation. Aliquots (25 μL) of a 0.25 mg/mL solution of γglobulins were placed into 1.5-mL microcentrifuge tubes and mixed with an equal volume of 2× SDS-PAGE sample buffer [0.0625 M Tris-HCl, pH 6.8, 15% (wt/vol) glycerol, 3.5% (wt/vol) SDS, 5% (wt/vol) 2-mercaptoethanol and 1.25% (wt/vol) bromophenol blue]. Controls were incubated for 3 min at either 37°C in a water bath or at 100°C in a heating block. As the negative control, 37°C was used (i.e., not boiled) in order to illustrate that the denaturation was as a result of boiling and not the effect of the sample buffer. Other samples were heated for various times from 10– 90 s in 10-s increments on high power in a domestic 800-W microwave oven with a turntable (Panasonic Model NN5452BBPQ). Microcentrifuge tubes were supported in a plastic “hedgehog” style test-tube rack (Nalge, Rochester, NY, USA) placed in the center of the oven. Molecular-weight-size markers and 20 μL of each sample were loaded onto a 10% SDS-PAGE mini-gel and electrophoresed at 150 V until the dye front reached the separating gel and then at 200 V until the dye front reached the bottom of the gel. The gel was stained with Coomassie blue and destained with acetic acid/methanol. Denaturation of γ-globulins by microwave irradiation is evident after 30 s and complete by 80 s (Figure 1). This is less than half the time typically used in other heat-denaturation procedures. Furthermore, only the liquid within the tube became hot, and sufficient pressure to pop open the lids was not produced. The tubes remained cool enough to be removed from the microwave oven by hand and very little evaporation was observed on the walls of the tubes.