Electrophoresis blue native

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. 

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. 

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. 

Mass spectrometric identilication of mitochondrial oxidative phosphorylation subunits separated by two-dimensional blue-native polyacrylamide gel electrophoresis. Electrophoresis 23, 2525-2533. 

Camacho-Carvajal, M.M., Wollscheid, B., Aebersold, R., Steimle, V. and Schamel, W.W. (2004) Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates a proteomics approach. Mol. Cell. Proteomics 3, 176—182. 

Devreese, B., Vanrobaeys, F., Smet, J., Van Beeumen, J. and Van Coster, R. (2002) Mass spectromet-ric identification of mitochondrial oxidative phosphorylation subunits separated by two-dimensional blue-native polyacrylamide gel electrophoresis. Electrophoresis 23, 2525-2533. 

Novakova, Z., Man, P., Novak, P., Hozak, P. andHodny, Z. (2006) Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis. Electrophoresis 27, 1277-1287. 

Another approach to overcoming the limitations inherent in the lEF dimension of 2-DE is to use alternative types of 2-D separations. 2-D blue native (BN) electrophoresis (Schagger and von Jagow, 1991) can be used to separate membrane and other functional protein complexes as intact, enzymatically active complexes in the first dimension. This is followed with a second-dimension separation by Tricine-SDS-PAGE to separate the complexes into their component subunits. This method, combined with protein identification by MALDI PMF, has been applied to several studies of the mitochondrial proteome (Brookes et al., 2002 Kraft et al., 2001). In a study of human heart mitochondria using BN/SDS-PAGE, the individual subunits of all five complexes of the oxidative phosphorylation system were represented and a novel variant of cytochrome c oxidase subunit Vic was reported (Devreese et al., 2002). 

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. 

Immunoprecipitation of urease or blue native gel electrophoresis established that urease apoenzyme is associated with the membrane protein, Urel, raising the possibility that the NH3 released during urease activity is able to diffuse rapidly through the inner membrane into the periplasm, decreasing the possibility of excessive alkalinization of the cytoplasm. 

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 ... 

Mailloux, R. J. Singh, R. Appanna, V. D. In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis. Anal. Biochem. 2006, 359, 210-215. 

J. Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE. Electrophoresis 2007, 28, 1036-1045. 

In the first dimension, 20 [tl plasma is separated by electrophoresis at 4°C in a 0.75% agarose gel using a 1 2 16 dilution of a barbital buffer. Bromophenol blue is added to a standard sample to visualize albumin in the native gel. The electrophoresis is stopped when the albumin/bromophenol blue marker has migrated 6 cm. Agarose gel strips containing the preseparated lipoproteins are then transferred to a 4-20% polyacrylamide gradient gel. Separation in the second dimension is performed at 40 mA for... 

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