2-D polyacrylamide gel electrophoresis

Figure 8.10 2-D polyacrylamide gel electrophoresis. Proteins from a lysate of Escher-...

Figure 4.2 Schematic overview of two protein identification strategies commonly followed in proteomics. Protein samples are separated by either two-dimensional (2-D) or one-dimensional (1 -D) polyacrylamide gel electrophoresis (PAGE). In both strategic tracks, proteins are converted into a set of peptides by enzymatic digestion (e.g., with trypsin) prior to MS analysis. Peptide mass fingerprinting (PMF) by MALDl MS is predomi-...

Figure 14 Chiral separation of dansyl amino acids by chiral polyacrylamide gel electrophoresis. Peak identification 1 = dansyl-L-glutamic acid, 2 = dansyl-D-glutamic acid, 3 = dansyl-L-serine, 4 = dansyl-D-serine, 5 = dansyl-L-leucine, 6 = dansyl-D-leu-cine. (From Guttman, A., Paulus, A., Cohen, A. S., Grinberg, N. and Karger, B. L., /. Chromatogr., 448, 41, 1988. With permission.)...

Fig. 10. A. Acetic acid-urea-triton-X-100 polyacrylamide gel electrophoresis [15] of the histones used to reconstitute 208-12 nucleosome arrays consisting of recombinant H2A.Z (lane 2) or recombinant H2A.1 (lane 3). Lanes 1 and 4 respectively are chicken erythrocyte and calf thymus histones used as markers [42]. B. Ionic strength (NaCl concentration) dependence of the average sedimentation coelRcient (s2o,w) of reconstituted 208-12 nucleosome arrays containing either H2A.1 (O) or H2A.Z ( ) [42]. The dotted line represents the behavior of a 208-12 complex reconstituted with chicken erythrocyte histones [406]. [Reproduced from Abbott D.W. et al. (2001) I. Biol. Chem. 276, 41945-41949, with permission from The American Society for Biochemistry and Molecular Biology.]...

Analysis by SDS-polyacrylamide gel electrophoresis of purified NOS from isolated HC from rats injected with killed Corynebacterium parvum (H), and from the murine macrophage cell line RAW 264-7 (M) (courtesy of D. Stuehr, The Cleveland Clinic, Cleveland, OH) which was exposed to LPS and IFNy. Crude cytosols were separated using ion exchange and affinity 2 5 -ADP Sepharose chromatography. Last step by gel filtration is equivalent to separation by molecular weight. 

To test this hypothesis, very low density lipoprotein (VLDL, d<1.0 gm/ml), low density lipoprotein (LDL, d=l.02-1.063) and high density lipoprotein (HDL, d=l.09-1.21) were isolated from outdated human plasma by ultracentrifugation according to established procedures (27,28), using potassium bromide for density adjustments and stored at -20° C in the presence of 20% sucrose before use. The purity of individual lipoprotein fractions thus obtained was established by polyacrylamide gel electrophoresis in sodium dodecyl buffer system (2 ) and filtration through a Sepha-rose 6B column, equilibrated with 0.2 M potassium bromide in 0.1 M sodium phosphate buffer, pH 7.2. Protein (30) and cholesterol... 

One of the most useful techniques for visualization of the proteome is two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE). This technique possesses unmatched resolving power for separation of proteins [2-4] and has been used extensively to analyze proteins [5-8], their regulation [9-18], and posttranslational modifications [19-22], Several tech-... 

Complex II consists of four polypeptide subunits (70,000, 27,000, 13,500, and 7,000 daltons). The two larger subunits are components of succinate dehydrogenase and this enzyme or the entire complex II was directly incorporated into phospholipid vesicles at the high protein lipid ratio of 1 2 (w/w). The phospholipid (egg yolk lecithin) contained a small amount of lipid (d) (Table 6.2 I molecule in 1000) or lipid (e) (Table 6.2 1 in 400). After irradiation the labeling pattern was analyzed directly by SDS-polyacrylamide gel electrophoresis. Presumably the lipids that did not become attached to polypeptides ran at the dye front, although this was not demonstrated. 

Significant improvements in the technologies of high-resolution two-dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE) and Mass Spectrometry (MS) have marked the start of proteome analysis. Proteomics permits the analysis of thousand of proteins simultaneously, and have the potential to identify markers for early detection, classification and prognosis of diseases, as well as pinpointing targets for improved treatment outcomes [42]. 

Sammons, D. W., Adams, L. D., and Nishizawa, E. E. (1981). Ultrasensitive silver-based color staining of polypeptides in polyacrylamide gels. Electrophoresis 2, 135-141. 

The Rotofor has been incorporated into two-dimensional purification schemes based on the principles of 2-D PAGE. Fractions collected from the Rotofor were further purified by 2-D PAGE62 or by preparative polyacrylamide gel electrophoresis.63-65 Highly purified proteins were obtained by this scheme, even low-abundance proteins, to allow for multiple analyses and for use as antigens. 

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