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The net charge of a protein is the sum of all negative and positive charges of the amino acid side chains; the three-dimensional configuration of the protein also plays a role. At low pH values, the carboxylic side groups of amino acids are neutral: R-COO- + H+ → R-COOH At high pH values, they are negatively charged: R-COOH + OH- → R-COO- + H2O The amino, imidazole and guanidine side-chains of amino acids are positively charged at low pH values: R-NH2 + H+ → R- NH3 + at high pH values, they are neutral: R-NH3

+ + OH- → R-NH2 + H2O For composite proteins such as glyco- or nucleoproteins, the net charge is also influenced by the sugar or the nucleic acid moieties. The degree of phosphorylation also has an influence on the net charge. Many of the microheterogeneities in IEF patterns are due to these modifications in the molecules. If the net charge of a protein is plotted versus the pH , a continuous curve which intersects the abscissa at the isoelectric point pI will result. The net charge curve is characteristic of a protein.

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Amphoteric buffers with a spectrum of isoelectric points between 3 and 10 form a pH gradient under the influence of the electric field. •  Mixtures of 600 to 700 individuals. •  High buffering capacities at their isoelectric points. •  2.5 % (w/v) in the gel.

• For traditional high resolution two-dimensional electrophoresis, carrier- ampholyte-IEF has mostly been performed in vertical individual gel rods (4-5 % T) in glass tubes.

(One-dimensional IEF is performed in thin horizontal gel layers on film supports: agarose or polyacrylamide. Preparative IEF can be performed in thick horizontal dextrane gel: Ultrodex.)

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1st phase. Stacking and concentration of sample components in an isotachophoresis system (because of discontinuity of ions) 2nd phase: Stack arrives at resolving gel (small pore size), which is highly restrictive to proteins. 2nd concentration effect. As the gel is not restrictive to glycine,. glycine ions pass proteins with constant speed: now proteins are in a homogeneous buffer. 3rd phase: Proteins are destacked and separated according to zone electrophoresis conditions. Advantages: • Slow migration in the beginning prevents protein aggregation while entering the gel matrix. • Two times concentration of zones lead to sharp bands and high resolution.

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Acrylamido buffers are copolymerized with the polyacrylamide network. A linear gradient has to be cast. 6 to 10 individuals (2 - 4 acids, 4 - 6 bases). They act in an acid-base titration way. •  Immobilines are nonamphoteric compounds, general structure: CH2=CH-CO-NH-R, defined by the pK values: R = weak acid, contains a carboxylic group, or R = weak base, contains a tertiary amino group. •  IPG gels are cast on film support mostly as 0.5 mm thin layers, washed with distilled water, dried down, and rehydrated before use with water or urea-detergent solution. All separations are run horizontally. Pharmacia Biotech Products, and Trademarks: • Immobiline II: Buffering acrylamide derivatives for casting IPG gels (Acrylamido buffers) • Immobiline DryPlate: Ready-made Immobiline gels washed and dried for isoelectric focusing in immobilized pH gradients. • Immobiline DryStrip: For high resolution two-dimensional electrophoresis, narrow strips (3 mm) are produced, which have no edge effects, because the gradient is fixed to the matrix.

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Membrane protein solubilization with nonionic detergents: these detergents would interfere with SDS. Thus SDS PAGE can not be used. Schägger, H. and von Jagow, G.: Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199 (1991) 223-231 Coomassie Brilliant Blue G-250 is added to cathodal buffer in native PAGE. • All proteins that bind Coomassie dye (all membrane proteins, some others) migrate to the anode at pH 7.5, also basic proteins (analogous to SDS) • Negatively charged proteins surfaces repel each other, this minimizes aggregation. • Negatively charged membrane proteins are soluble in detergent-ffree solution. • Proteins migrate as blue bands through the gel.

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