Polyacrylamide Gel Electrophoresis Systems

Other supports in electrophoresis are agarose gels, paper, or celluloses acetate. 

If proteins are in contact with some detergents as sodium do-decylsulfate (SDS) or cetyltrimethylammonium bromide (CTAB) they become denaturated, e.g., their secondary, tertiary, and quaternary structures are destroyed. They get a rod-like shape and the amount of bound detergent is proportionate to the molar mass of the proteins These protein-detergent complexes have negative charges at slightly alkaline pH if SDS is used and their size (hydrodynamic radius) is approximately proportional to their molar 

The relative mobihty of a macromolecule is given by the quotient of its distance of migration measured from the start of separation and the distance of electrophoresis front (position of tracking dye)  

If Ry of different proteins should be compared, the calculations have to be made from the same slab gel to eliminate variances in acrylamide concentration, polymerization, and electrophoretic conditions. 

The gel composition is often described by the terms %T and %C. %T refers to the total content of acrylamide (sum of acrylamide and cross-linking monomer), whereas %C is the part of cross-linking substance (e.g., N,N -methylene bisacrylamide) of monomers. 

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Hl-trap Benzamidine EE column SDS-polyacrylamIde gel electrophoresis system Thrombin BOO units In 0.5 ml PBS (stored at -80°C) Eactor Xa 400 units In water to give a final solution of 1 Unlt/pil stored at-80°C... 

Sterile microcentrifuge tubes Sterile pipettes and pipette tips SDS-polyacrylamIde gel electrophoresis system... 

Macfarlane, D.E. (1983) Use of benzyldimethyl-n-hexadecylammonium chloride ( 16-BAC ), a cationic detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells. Anal. Biochem. J32, 231-235. 

The fourth FMD virus capsid polypeptide translated has been designated VP3, VPl, or VPthr, due to its variable migration in different polyacrylamide gel electrophoresis systems. This protein will be referred to as VPl as recommended at the 1982 American Society of Virology meeting at Ithaca, NY. 

The cis-trans isomerase of 16 l(9c) (9-Iase), which locates in the cytosolic fraction [3], was purified by ammonium sulfate precipitaion and column chromatography. At the final step the 9-Iase was 5340 times purified and its recovery of activity was 5%. The purified enzyme preparation exhibited the sole band with a molecular mass of 80 kDa on incompletely-denaturing and denaturing polyacrylamide gel electrophoresis systems, indicating that the 9-Iase is a monomeric protein with a molecular mass of 80 kDa. 

Polyacrylamide gel electrophoresis is one of the most commonly used electrophoretic methods. AnalyMcal uses of this technique center around protein characterization, for example, purity, size, or molecular weight, and composition of a protein. Polyacrylamide gels can be used in both reduced and nonreduced systems as weU as in combination with discontinuous and ief systems (39). 

Jackson, P., The use of polyacrylamide-gel electrophoresis for the high-reso-lution separation of reducing saccharides labeled with the fluorophore 8-ami-nonaphthalene-l,3,6-trisulfonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device, Biochem. ]., 270, 705, 1990. 

Poehling reported a microscale two-dimensional gel electrophoresis system 25 years ago (Poehling and Neuhogg, 1980 Neuhoff, 2000). In that system, separation in the IEF dimension was performed in thin, gel-filled tubes. After IEF, the gel was transferred to a 3 cm x 3.5 cm polyacrylamide gel, where proteins were separated by SDS-PAGE. Several hundred components were resolved from a few micrograms of protein homogenate. 

Electrophoretic Methods Systems for polyacrylamide gel electrophoresis, 104, 237 preparative isoelectric focusing,104, 256 gel... 

We have examined whether the sulfur that was bound to the proteins of a reconstituted system from the liver of phenobarbital-treated rats was bound to both the reductase and cytochrome P-450. I this experiment, the reconstitued system was incubated with [ s] parathion. The reaction mixture was dialyzed and applied to a Sephadex G-25 column to remove the last traces of unreacted parathion and its noncovalently bound metabolites. The protein fraction from the Sephadex column was reduced in volume and subjected to SDS-polyacrylamide gel electrophoresis in the absence of either dithiothreitol or mercaptoethanol. The results are shown in Figure 5. There was considerable protein and radioactivity at the origin. This material at the origin represents an aggregate of reductase... 

A fully automated two-dimensional electrophoresis (2DE) system for rapid and reproducible protein analysis is described. 2DE that is a combination of isoelectric focusing (lEE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein expression analysis. Here, all the operations are achieved in a shorter time and all the transferring procedures are performed automatically. The system completed the entire process within 1.5 h. A device configuration, operational procedure, and data analysis are described using this system. 

SDS-polyacrylamide gel electrophoresis Protein samples were analyzed under denaturing conditions in a discontinuous gel system as described by Laemmli [13] using a 5% stacking gel and a 12% separating gel in a vertical gel system (BioRad, Mini Protean 11, CA, USA). 

Wyckoff, M., Rodbard, D. and Chrambach, A. 1977. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffer using multiphasic buffer systems Properties of the stack, valid Rf measurements, and optimized procedure. Anal Biochem. 78, 459-482. 

Figure 8-14 SDS-polyacrylamide gel electrophoresis of human erythrocyte ghosts. (A) From untreated cells. (B) From cells digested externally with chymotrypsin. (C) Inside-out vesicles prepared from cells pretreated with chymotrypsin. (D) The same inside-out vesicles after further treatment with chymotrypsin. (E) Polypeptides released hy the chymotryptic treatment of the inside-out vesides. The peptides are numbered according to the system of Steck232 Hb, hemoglobin. From Luna et al233...

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