Native electrophoresis

Wittig, I., Karas, M., and Schagger, H. (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol. Cell. Proteomics 6, 1215-1225. 

The choice of native electrophoresis system depends on the particular proteins of interest. There is no universal buffer system ideal for the electrophoresis of all native proteins. Both protein stability and resolution are important considerations in buffer selection. Recommended choices are the Omstein-Davis discontinuous system21-24 and McLellan s continuous buffers.25... 

Other buffers that have been used for continuous, native electrophoresis are Tris-glycine (pH range 8.3 to 9.5),19 Tris-borate (pH range 8.3 to 9.3),26 and Tris-acetate (pH range 7.2 to 8.5).27 Borate ions26 can form complexes with some sugars and can therefore influence resolution of some glycoproteins. 

Darley-Usmar, V. M. (2002). ffigh throughput two-dimensional blue-native electrophoresis a tool for functional proteomics of mitochondria and signaling complexes. Proteomics 2, 969-977. 

Schagger, H. and von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. 

An important and often overlooked variegation of zone electrophoresis-based separations is the mixed native-denaturing two-dimensional (2D) electrophoresis developed mainly by Schagger and coworkers (Schagger and Von Jagow 1991). Because the first dimension is a native electrophoresis, this approach provides invaluable information upon the assembly of membrane complexes. However, it poses... 

Nijtmans, L.G., Henderson, N.S. and Holt, I.J. (2002) Blue Native electrophoresis to study mitochondrial and other protein complexes. Methods 26, 327-334. 

Electrokinetic methods certainly comprise some of the most powerful techniques for protein analysis. They are based on the property of charged molecules to migrate in an electric field according to their net charge (isoelectric focusing, IEF), net charge and size (native electrophoresis) or only size (SDS-PAGE). These techniques include both analytical and preparative systems. 

Figure 7.4 Native electrophoresis of proteins from different grape varieties (Reproduced from J. Agric. Food Chem., 1999, 47(1), 114-120, Moreno-Arribas et al., with permission from the American Chemical Society)...

Measurements of total tyrosinase activity do not reflect its isoenzyme composition. Therefore, we examined the isoenzyme forms of tyrosinase in small pins, large pins, immature and mature mushrooms by native electrophoresis followed by staining for dopa oxidase activity. Using dopa oxidase as an indicator of tyrosinase, one dominant isoenzyme form (I) was present in all development stages (Fig. 3). Two faster migrating forms (IIIa,b) were also apparent as well as a faint intermediate migrating form (II). No new isoenzyme forms were observed during development. 

Figure 3. Extracts prepared in the absence (-) and presence (+) of phenolic adsorbents were subjected to native electrophoresis and stained for tyrosinase using dopa (A). Western blot of mushroom tyrosinase in four developmental stages after SDS PAGE (B). Samples were the same as in A (Top photograph reproduced with permission from ref. 35. Copyright 1989/ Food ScL)...

The above samples in each break were analyzed for tyrosinase isoenzymes by native electrophoresis to determine if any changes in isoenzyme forms occurred from break to break (Fig. 5). All samples applied to electrophoresis contained similar amounts of protein (15 ug). One dominant slower form (I) was present in all three breaks. The intensity of this form appeared to decrease with development in each break. Two faster migrating forms (III a,b) were also observed in each break and these two forms also decreased in intensity with development. No apparent trend was noted in the amount of the intermediate migrating forms (II). These profiles were qualitatively similar to fresh mushroom extract profiles at different developmental stages and once again suggest that no new isoenzyme forms were made during development or in different breaks. 

Figure 5. Tyrosinase isoenzyme forms identified in three different breaks (1—3) after native electrophoresis and staining for dopa oxidase activity. A—D represent small pins, large pins, immature, and mature mushroom samples, respectively. (Reproduced with permission from ref. 35. Copyright 1989/. Food ScL)...

Interestingly, staining of native electrophoresis gels of P. rhodozyma cell extracts indicated that only the mitochondrial Mn-SOD is present, and that the yeast apparently lacks the cytosolic Fe- and Cu-forms of SOD (31). Catalase activity was also present in low quantities in P. rhodozyma compared to Saccharomyces cerevisiae. These observation suggested that carotenoids may partly compensate for the lack of SOD and low activity of catalase in the cytosol. 

Schagger, H., Cramer, W. A, and von Jagow, G. (1994). Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal. Biochem. 217, 220-230. 

The conditions of electrophoresis are most important for success of the SSCP technique. Separation of ssDNA by nondenaturating (native) electrophoresis may be performed either in slab gels (native PAGE) or in capillaries (CE). The decision for one of these methods may depend on the equipment available and the number of samples to be analyzed. 

Problems Serva Blue G presumably prefers to attach to trans-membrane regions. Large membrane proteins thus show a lesser charge density than small membrane proteins. Soluble proteins bind still less stain. The charge-to-mass ratio of different protein-stain complexes is thus not constant, and the native electrophoresis does not separate the protein complexes by MW (personal communication by A. Schrattenholz, Mainz). Soluble marker proteins such as thyroglobulin, ferritin, and the like smudge in the gel or partially disintegrate into subimits. 

Native electrophoresis of pepsin and hemoglobin on 10% polyacrylamide gel carried out at 48 °C during 90 min, according to the Laemmli procedure, at pH 8.3 (Laemly 1970). Water solutions of all samples of enzyme (pepsin dissolved in water to final concentration of 2 mg/mL) were titrated with HCl to pH 2 and incubated at 37 °C, with addition of different concentrations of Al + ion (1, 5 and 10 mM). The samples were diluted with sample buffer in ratio 1 1 (v/ v) and applied on gel in volume of 20 pL. Visualization was performed with Commassie Brilliant Blue G-250 dye. The gels scanned and processed using Corel Draw 11.0 software package. Quantification of electrophoretic mobility of the molecule is carried out via Rs value, where it is defined by ... 

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