Gel electrophoresis in the presence of SDS

SDS dissociates most proteins and reacts with them so as to equalize their charge properties. The electrophoresis protocols for SDS-pro-teins are, hence, not as diverse as for native proteins. 

The continuous SDS-buffer of Shapiro et al. (1967) and Weber and Osborn (1969) is widely used. In this system the same phosphate buffer (100 mM, pH 7.0) is used, both for the gels and the electrode compartments. Protein separation under such conditions is consequently slow. The discontinuous system of Laemmli (1970), adapted from the system earlier described by Davis (1964) and Ornstein (1964), produces considerably faster separation than the continuous system. 

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SBA, prepared as described herein, possessed an ultraviolet (UV) absorption spectrum and an extinction coefficient comparable to those previously reported (5). Polyacrylamide gel electrophoresis in the presence of SDS resolved SBA into two closely spaced peptide bands comparable to those reported by Lotan et al. (6). ... 

In the field of research methods probably the most important application of protein surfactant complexation is in the technique of polyacrylamide gel electrophoresis in the presence of SDS, the so-called SDS-PAGE technique, used for the analysis and estimation of molecular masses of protein subunits [22], Protein subunit-SDS complexes are formed from proteins reduced by /3-mercaptoethanol to remove disulphide bonds. The binding of SDS to the polypeptide chains oc-... 

Subunit Composition of a Protein A protein has a molecular mass of 400 kDa when measured by gel filtration. When subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), the protein gives three bands with molecular masses of 180, 160, and 60 kDa. When electrophoresis is carried out in the presence of SDS and dithiothreitol, three bands are again formed, this time with molecular masses of 160, 90, and 60 kDa. Determine the subunit composition of the protein. 

An excess amount of surfactant can solubilize proteins, presumably by additional adsorption to the surface and generation of a large net charge at the surface of the protein molecule. This is the basis for estimation of protein molecular weight by gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). Such effects are usually found only at surfactant concentrations well in excess of those normally found in foods, and have more use in laboratory investigations than in ordinary applications. 

Some argument remained until recently to cast doubt on the number of subunit types in the MoFe protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) on Cpl (Huang et al., 1973) and Kpl (Eady et ai, 1972) indicated the presence of two subunit types. However, Bulen (1976) cast doubt upon the interpretation of results obtained with SDS-PAGE when he showed that carboxymethylated Avl migrated as a single band whereas the untreated protein showed two types of subunits. Bulen attributed the appearance of two types of subunits to Fe associated... 

Abbreviations used Fi Fq, H+-translocating ATP synthases from mitochondria, E. coli plasma membranes, plasma membranes of the thermophilic bacterium PS3 and chloroplast membranes CFi, chloroplast coupling factor one ATPase, CFiE. C. 3.6.1.3 SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl... 

To analyze the protein products of 29 genes a culture of B. subtilis, irradiated with UV-light to inhibit host protein synthesis, is infected with 29 in the presence of radioactive amino acids. At the end of the labeling period the cells are harvested, lysed with lysozyme and subjected to polyacrylamide slab gel electrophoresis in the presence of the detergent sodium dodecylsulfate (SDS). After electrophoresis the gel slabs are dried and autoradiographed to determine the positions of the labeled proteins. This system of electrophoresis resolves the protein chains on the basis of size and allows an estimate of the molecular weight of each protein band. 

Figure 3. SDS-PAGE and in situ pectinase activity on pectin and polygalacturonic acid-agarose overlays of culture filtrates of Aspergillus niger N-402 (upper panel) and Aspergillus FP-180 (lower panel) at 2.5, 3.5, 5.5 and 6.5 pHi (Lanes a, b, c, and d, respectively). Electrophoresis on 10% acrylamide slab gel (14 X 8 cm) in the presence of SDS was according to Laemmli (6), run at 30 mA constant current for 2 hours. Crude cell-free samples were concentrated by lyophilization, dialyzed, boiled with sample buffer by 60 sec. and applied to each well. Polyacrylamide gel and overlays were incubated overnight with 0.17 acetate buffer at room temperature.

Figure 7.1. Effect of GM-CSF on neutrophil protein biosynthesis. Human neutrophils were incubated in RPMI1640 medium supplemented with 2.5% foetal calf serum and 60 jtCi/ml [35S]-methionine, in the presence and absence of 50 U/ml GM-CSF. After 4 h incubation at 37 °C, the cells were pelleted by low-speed centrifugation. The proteins in the cell pellets were precipitated by 10% trichloracetic acid and then analysed by two-dimensional polyacrylamide gel electrophoresis, using iso-electrofocussing in the first dimension. Electrophoresis in the second dimension was performed in the presence of SDS and used a 12% acrylamide gel. Source Experiment of Becky Stringer.

A few practical notes should be emphasized regarding SDS electrophoresis. Potassium salts should be avoided and sodium salts used in their places because potassium dodecyl sulfate is quite insoluble. In addition, even sodium dodecyl sulfate is insoluble below about 10°C. Although not specifically cited above, disc gel electrophoresis may also be used in the presence of SDS. These techniques, described by Studier (11) and Ames (12), are of great advantage when the sample volume is in excess of 10 to 20 /Ltliters per gel. A widely used buffer system for SDS acrylamide gel electrophoresis is that developed by Laemmli (41). 

The membrane-bound H. saccharovorum ATPase can be purified to a specific activity of 3.6(imol Pj/min/mg protein [46]. The native enzyme (Mr 350000) is composed of four subunits. Molecular-mass determinations in the presence of sodium dodecylsulfate (SDS) provide Mr estimates of 87 000, 60 000, 29 000, and 20 000 for the subunits, designated I, II, III, and ly respectively [23], SDS gel electrophoresis overestimates the molecular masses of acidic proteins [70] and more accurate values can be obtained by electrophoresis in the presence of cetyltrimethylammonium bromide [71]. When this is done, the resulting values for Mr agree with the those obtained using SDS [46]. 

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