Native polyacrylamide gel electrophoresis

Analyses by native polyacrylamide gel electrophoresis and circular dichroism (CD) spectroscopy revealed that spontaneous coiled-coil associations between EGF-E5-His and EGF-K5-His promoted heterodimer (dEGF-His) formation. The CD spectroscopic analysis suggested that the E5 peptide in monomeric EGF-E5-His had a disordered structure. However, the ot-helical structure was induced in the E5 peptide when it associated with EGF-K5-His. These findings are shown schematically in Fig. 6. 

Fig. 1. Native polyacrylamide gel electrophoresis. The protein samples are loaded into the sample wells formed in the top of the gel. An electric field is applied across the gel from top to bottom and the proteins migrate down through the gel. The smaller the protein and the greater its net negative charge, the further it will migrate.

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

Fig. 7. Oxidative refolding of reduced RNase Tl. Reoxidation conditions were 0.1 M Tris-HCl, pH 7.8, 0.2 Af guanidinium chloride, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 0.2 mM EDTA, and 2.5 nM RNase Tl at 25°C. The kinetics of oxidative refolding were followed by the increase in tryptophan fluorescence intensity at 320 nm ( ), by an unfolding assay (Kiefhaber el ai, 1990b) that measures the formation of native protein molecules (A), and by the increase in the intensity of the band for native RNase Tl in native polyacrylamide gel electrophoresis ( ). Fluorescence emission in the presence of 10 mM reduced dithioerythritol to block disulfide bond formation (O). The small decrease in signal after several hours is caused by slight aggregation of the reduced and unfolded protein. (From Schonbrunner and Schmid (1992).

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. 

In order to identify the type of SOD after separation by native polyacrylamide gel electrophoresis, 2 mM KCN and 4 mM HjOj can be added to the staining mixture to study the inhibition pattern. 

SSCP is based on the conformation-dependent migration of single-stranded DNA (ssDNA) or RNA in native polyacrylamide gel electrophoresis (PAGE) or capillary electrophoresis (CE). The protocol of SSCP analysis used most commonly comprises the following steps (Figure 5.1) ... 

In another example, a hydrophilic star block copolymer was composed of a hyperbranched PEI core, a PLL inner shell and a PEG outer shell. Insulin, as a model protein, can be rapidly and efficiently encapsulated by the synthesized polymer in aqueous phosphate buffer at physiological pH. Complexation between PEI-PLL-Z)-PEG and insulin was demonstrated using native polyacrylamide gel electrophoresis. An in vitro release study by dialysis showed sustained release of the encapsulated protein at physiological pH, and an accelerated release when the pH was decreased. The insulin released from the star block copolymer retained its chemical integrity and immu-nogenicity. Successful in vitro uptake studies of enhanced green fluorescent protein into Ad293 cells mediated by PEI-PLL-Z)-PEG were also performed. ... 

PPase activity was measured as described in (11). Native polyacrylamide gel electrophoresis was carried out by the procedure described in (12). The activity staining of the gel for PPase was performed as follows. To localize the PPase activity the gel was incubated for 30 min at room temperature in a solution identical with that utilized... 

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. 

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