ARTICLE continued from page 9 pensive method to maintain very low basal may be observed in the absence of glucose. tionary phase, we recommend the addition expression levels of T7 RNA polymerase in Overall, the optimal combination of strin- of 0.5 to 1.0% glucose to the medium, so the λDE3 lysogenic expression hosts used gent uninduced repression and high in- that the glucose effect can be exploited to in the pET System. This is especially true duced expression may be achieved by initial reduce basal expression. when λDE3 hosts carrying pET plasmids growth in the presence of glucose, followed are grown to stationary phase. A disadvan- by switching to medium without glucose tage with glucose addition is that after an for induction. REFERENCES initial rapid growth phase the metabolic Novagen’s recommendations for growth 1. Grossman, T. H., Kawasaki, E. S., breakdown products of glucose will lead to and induction of pET constructs in expres- Punreddy, S. R., and Osburne, M. S. acidic culture conditions and lower cell sion hosts are based on the information pre- (1998) Gene 209, 95–103 density at stationary phase. The data pre- sented above. For innocuous proteins, any 2. Pan, S. and Malcolm, B. A. (2000) sented in Figure 2 demonstrate that strong pET vector and λDE3 lysogen are suitable BioTechniques 29, 1234–1238. induction can be achieved from λDE3 lyso- in a variety of media. But for proteins that 3. Le Grice, S. F. J., Matzura, H., Marcoli, gens in the presence of glucose for some are potentially toxic to the bacterial cell, we R., Iida, S., and Bickle, T. A. (1982) J. Bacteriol. 150, 312–318. target proteins. Note, however, that theoret- recommend using either a pET vector with 4. Kelley, K. C., Huestis, K. J., Austen, D. ically the strongest induction of T7 RNA a T7lac promoter or expression hosts that A., Sanderson, C. T., Donoghue, M. polymerase would be expected when glu- carry the pLysS plasmid. In addition, our A., Stickel, S. K., Kawasaki, E. S., and cose is absent and cAMP levels are elevated. general advice is to avoid growing a λDE3 Osburne, M. S. (1995) Gene 15, Accordingly, in some cases (see preceding lysogen carrying a pET plasmid to station- 33–36. article), higher target protein expression ary phase. If the cells must be grown to sta- Preparation of protein samples for SDS-polyacrylamide gel electrophoresis: procedures and tips Anthony C. Grabski1 and Richard R. Burgess2—1Novagen, Inc. and 2McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706 odium dodecyl sulfate polyacry- components because the proteins are fo- dious gel casting, buffer preparation and ap- lamide gel electrophoresis (SDS- cused, or “stacked,” as thin bands prior to paratus set-up, but on careful sample prepa- PAGE) is the most widely used ana- entering the resolving gel. Second was the ration and treatment prior to loading the lytical method to resolve separate use of the detergent sodium dodecyl sulfate gel. This article describes techniques and Scomponents of a protein mixture. It is (SDS) and reducing agents to denature pro- procedures as a guide for preparation of pro- almost obligatory to assess the purity of a teins (7). SDS binds strongly to proteins at tein samples for SDS-PAGE analysis. protein through an electrophoretic method. an approximate ratio of 1 dodecyl sulfate Sample buffer preparation SDS-PAGE simultaneously exploits differ- molecule per 2 amino acid residues (8). ences in molecular size to resolve proteins Therefore, the negative charge/unit mass To ensure consistent and successful differing by as little as 1% in their elec- ratio when SDS is bound to the polypeptide PAGE analysis, the highest purity reagents trophoretic mobility through the gel matrix chain is similar for all proteins. Third was should be used to prepare sample buffer (1). The technique is also a powerful tool for the combination of the first two discoveries stock solutions. After a reliable source of estimating the molecular weights of proteins employing a simple Tris-glycine buffer electrophoresis reagents has been identified, (2, 3). The success of SDS-PAGE as an in- system (9). More recently, buffer combina- the vendor and buffer component chemicals dispensable tool in protein analysis has been tions such as Tris-borate (10) and Tris- should be maintained. High purity elec- attributed to three innovations that permit- tricine (11) have improved the resolving trophoresis, Ultrol® grade, and molecular bi- ted the correlation of electrophoretic mobil- power of the original methods. Modern ology grade reagents are available through ity with a protein’s molecular mass (4). First SDS-PAGE has evolved to use microslab Novagen’s partner brand, Calbiochem. was the introduction of discontinuous buffer precast gels (12). Precast and packaged gels Solutions must be carefully and safely pre- systems where the sample and gel running in a wide variety of gel formulations, acry- pared, dated, and chemical lot numbers buffers differ in both composition, Tris- lamide percentages, thicknesses, well for- recorded. Concentrated stock solutions HCl/Tris-glycine, and pH, 6.8/8.3, respec- mats, and buffer systems are now commer- should not be stored for long periods of tively (5, 6). Discontinuous buffer systems cially available from several manufacturers. time. Tris base, rather than Tris-Cl, should allow larger sample volumes to be loaded Therefore, successful SDS-PAGE analysis of be used for buffer preparation and pH ad- while maintaining good resolution of sample protein samples no longer depends on te- justment made with HCl. Use of Tris-Cl inNovations 13 10 will result in a higher ionic strength, poor Table 1. SDS-PAGE sample buffer recipes 1.4 µg SDS per 1.0 µg polypeptide, but a migration and diffuse protein bands (13). A ratio of 3:1 is recommended (15). The 2X Component Concentration respirator or dust mask should be worn sample buffer prepared as shown in Table 1 2X 4X when handling powdered SDS. Sulfhydryl Tris-HCl, pH 6.81 0.125 M 0.25 M contains 40 µg/µl SDS. Maintained reduc- reagents, dithiothreitol (DTT) and 2-mer- SDS 4% 8% tion of protein sulfhydryls is essential in captoethanol (2-ME) can be unstable in so- 2-ME2 5% 10% order to prevent intramolecular disulfide lution and are toxic. These chemicals should DTT3 0.15 M 0.3 M bond formation through oxidized cysteines. be measured in a fume hood while wearing Glycerol 20% 30% If artifactual band heterogeneity or unusual gloves and safety glasses. Although 2-ME is Bromphenol blue .01% .02% doublets are noted in SDS-PAGE results historically the chemical of choice for reduc- 1. Prepared using Tris base, pH adjusted with HCl. from samples containing sulfhydryls, insuf- tion of protein disulfide bonds in SDS- 2. If 2-ME is used, omit DTT. ficient reducing agent was present during PAGE (7, 9), DTT is also a very effective al- 3. If DTT is used, omit 2-ME. sample treatment or the oxidation of cys- ternative (14). Glycerol is added to increase (BCA) assay prior to sample buffer addition. teines may have occurred during the stack- the sample density, facilitating gel loading Loading too much protein will result in dis- ing phase of electrophoresis. These artifacts and preventing convective migration out of torted, poorly resolved bands in the over- may be prevented if samples are treated with the sample wells. A small amount of loaded lane and distorted electrophoretic iodoacetamide (IAA) after heating in the ap- bromphenol blue is added as a visual aid patterns in adjacent lanes. Underloading propriate concentration of sample buffer. during sample loading and as a tracking dye, simply prevents detection of minor compo- The IAA treatment irreversibly blocks allowing easy monitoring of electrophoretic nents while even major bands will be too sulfhydryls and destroys excess reducing progress. faint for photographic reproduction of the agent (16). Therefore, the sample buffer The sample buffer recipes listed in Table recipes in Table 1 do not necessarily indicate 1 are commonly used for Tris-glycine SDS- functional dilution factors, but rather they PAGE analysis of protein samples under de- are convenient stock concentrations permit- naturing, reduced conditions (7, 9, 13). ting correct addition of reagents to samples When preparing these buffers, wear gloves of high and low protein concentration. to avoid keratin (skin protein) contamina- Delayed heating of samples after sample tion. A heterogeneous cluster of bands buffer addition or excessive heating can around 55 kDa can be seen when a keratin- cause electrophoretic artifacts due to protein contaminated sample or sample buffer is degradation and peptide bond cleavage, re- used. This is particularly obvious with high spectively. Upon addition of SDS sample sensitivity silver staining methods. The buffer, samples should be immediately sample buffer should be divided into 1-ml Sample preparation is critical mixed and heated to 85°C for three min- aliquots and can be stored frozen (–70°C) for clear and accurate resolution utes. This treatment is usually sufficient to for several months. Prior to use, warm of protein bands. reduce disulfides, solubilize and dissociate (37°C) and mix the solution briefly to com- proteins without peptide bond cleavage. pletely dissolve the SDS. gel. Depending on the well size and gel Addition of SDS sample buffer will begin to thickness, the amount of protein loaded denature most proteins. However, proteases Protein sample preparation should range from 0.5–4.0 µg for purified are known to be resistant to SDS denatura- Sample preparation is critical for clear samples and from 40–60 µg for crude sam- tion alone (15, 17). Partially denatured sam- and accurate resolution of protein bands. ples if a Coomassie blue stain (e.g., ples (particularly crude extracts) are there- Photographic quality results are routinely RAPIDstain™) is used.