Electrophoresis separated proteins

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

Electrophoresis separates proteins on the basis of mass or charge. SDS gel electrophoresis and isoelectric focusing can be used separately or in combination for higher resolution. 

I Gel electrophoresis is the best way to analyze mix-Vr-j tures and assess purity. Gel electrophoresis separates proteins according to their size and their charge. It is almost always performed in aqueous solution supported by a gel system. The gel is a loosely cross-linked network that functions to stabilize the protein boundaries between the protein and the solvent, both during and after electrophoresis, so that they may be stained or otherwise manipulated. 

Note that electrophoresis separates proteins on the basis of their net charge. If the electrophoretic picture shows only one protein component, and this is observed at several pH values, it may be suspected that the protein is homogeneous. To establish homogeneity more firmly, the protein should also be subjected to a procedure that analyzes it according to size, for example ultracentrifugation and/or electrophoresis in a polyacrylamide gel in the presence of urea and sodium dodecyl sulfate. 

Joubert, R., Strub, J.-M., Zugmeyer, S., Kobi, D., Carte, N., Van Dorsselaer, A., Boucherie, H., and Jaquet-Gutfreund, L. 2001. Identification by mass spectrometry of two-dimensional gel electrophoresis-separated proteins extracted from lager brewing yeasts. Electrophoresis 22, 2969-2982. 

Gel electrophoresis separates proteins on the basis of their rates of movement in an applied electric field. SDS-polyacrylamlde gel electrophoresis can resolve polypeptide chains differing In molecular weight by 10 percent or less (see Figure 3-32). 

Polyacrylamide gel electrophoresis separates proteins on the basis of size. [3-mercap-toethanol reduces the disulfide bonds that link the light and heavy antibody chains. Thus, the light and heavy chains are separated from one another on the gel. 

M. Galvani, E. Bordini, C. Peubelli, and M. Hamdan, Effect of experimental conditions on the analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis separated proteins by matrix-assisted laser desorption/ionisation mass spectrometry. Rapid Commun. Mass Spectrom. 14, 18-25 (2000). 

Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis separates proteins primarily on the basis of their molecular weights and is an extremely useful technique for analyzing membrane proteins. When the outer membrane is analyzed by SDS polyacrylamide gel electrophoresis, four major protein peaks are detected, as opposed to the numerous peaks observed in the case of the cytoplasmic membrane. The exact total number of protein components in the outer membrane has not yet been established. 

Guttman, A, On the Separation Mechanism of Capillary Sodium Dodecyl Sulfate-Gel Electrophoresis of Proteins, Electrophoresis 16, 611, 1995. 

SDS gel electrophoresis separation in total denaturing conditions was carried out on the protein of culture filtrates and proteins of known molecular mass. The four dark bands (Figure 2) which appear in the gel between 45 and 36 kDa of the standards were assumed to be PG based on the gel filtration results for PG activity and total protein. The relative molecular mass of the four protein bands were estimated as 45 kDa, 42 kDa, 39 kDa and 36 kDa. It was calculated that about 85% of total protein secreted into the culture medium by K. marxianus consisted of PG. 

Kato, Y., Nakamura, K., Yamazaki, Y., and Hashimoto, T., Comparison of high-performance ion-exchange chromatography and gel electrophoresis in protein separations, ]. Chromatogr., 318, 358, 1985. 

Figure 17 SDS-capillary gel electrophoresis of protein standards. Separation conditions 27 cm x 50 pm i.d. uncoated capillary 888 V/cm gel eCAP 200. (From Bene-dek, K. and Guttman, A., ]. Chromatogr., 680, 375, 1994. With permission.)...

A. Gerstner, Z. Csapo, M. Sasvari-Szekely, and A. Guttman, Ultrathin sodium dodecyl sulfate gel electrophoresis of proteins Effect of gel composition and temperature on the separation of sodium dodecyl sulfate-protein complexes, Electrophoresis, 21, 834 (2000). 

Figure 2.1. Schematic illustration oftwo-dimensional gel electrophoresis. Proteins are extracted from the organism of interest and solubilized. The first dimension separates proteins based on isoelectric point. The pi strip is reduced and alkylated and applied to an SDS-PAGE gel for separation by molecular weight. Proteins canbe visualized using a number of staining techniques.

Pietrogrande, M.C., Marchetti, N., Dondi, F., Righetti, P.G. (2002). Spot overlapping in two-dimensional polyacrylamide gel electrophoresis separations a statistical study of complex protein maps. Electrophoresis 23, 283. 

Hoffmann, R, Ji, H., Moritz, R. L., Connolly, L. M., Frecklington, D. F., Layton, M. J., Eddes, J. S., Simpson, R. J. (2001). Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215 a non two-dimensional gel electrophoresis-based proteome analysis strategy. Proteomics 1(7), 807. 

There has been interest in miniaturizing and automating electrophoresis of proteins. Ruchel (1997) reported a miniaturized system where proteins are first separated by isoelectric focusing in millimeter diameter tubes. The tube s contents are transferred to a slab gel that is a few centimeters on a side. This technology was used to separate the proteins from a single giant neuron from Aplasia califomicus. More recently, native fluorescence has been used to resolve 200 proteins from a similar miniaturized electrophoresis system (Sluszny and Yeung, 2004). 

FIGURE 15.1 One-dimensional capillary electrophoresis separation of a protein homogenate prepared from the hTERT cell line. Both separations were preformed in 30 pm ID, 145 pm OD, 20 cm long capillaries at 20,000 V. (a) Micellar electrokinetic chromatography performed with a 100 mM CHES, 100 mM Tris, and 15 mM SDS buffer at pH 8.7. Sample is electro-kinetically injected with 0.25 kV for 1 s (b) Capillary sieving electrophoresis performed in 5% Dextran (513 kDa), 100 mM CHES, 100 mM Tris, 3.5 mM SDS, pH 8.7. 

Issaq, H.J., Chan, K.C., Cheng, S.L., Qingho, L. (2001). Multidimensional high performance liquid chromatography-capillary electrophoresis separation of a protein digest an update. Electrophoresis 22, 1133-1135. 

Seow TK et al. Two-dimensional electrophoresis map of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated proteins by... 

Various methods have been used to examine the composition of proteins adsorbed to SAMs. Overall adsorption patterns can be examined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [50, 76, 77]. Absorbed proteins are eluted from the surface with surfactant (SDS), and then separated by electrophoresis. The proteins of interest are examined by western blotting [50, 76, 77]. Protein-specific antibodies can be used to detect proteins of... 

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