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1. Illustration of electrophoresis. 2. Illustration of electrophoresis retardation. Electrophoresis is the motion of charged dispersed particles or dissolved charged molecules relative to a fluid under the influence of a spatially uniform electric field.
There are limits to electrophoretic techniques. Since passing a current through a gel causes heating, gels may melt during electrophoresis. Electrophoresis is performed in buffer solutions to reduce pH changes due to the electric field, which is important because the charge of DNA and RNA depends on pH, but running for too long can exhaust the ...
Anionic dyes of a known electrophoretic mobility are usually included in the sample buffer. A very common tracking dye is Bromophenol blue. This dye is coloured at alkali and neutral pH and is a small negatively charged molecule that moves towards the anode. Being a highly mobile molecule it moves ahead of most proteins. [20]
The electrophoretic linear (horizontal) separation of proteins by Ip along a pH gradient in a polyacrylamide gel (also known as isoelectric focusing), followed by a standard molecular weight linear (vertical) separation in a second polyacrylamide gel , constitutes the so called two-dimensional gel electrophoresis or PAGE 2D. This technique ...
Proteins are separated by both electrical forces and electroendoosmostic forces. The net charge on a protein is based on the sum charge of its amino acids, and the pH of the buffer. Proteins are applied to a solid matrix such as an agarose gel, or a cellulose acetate membrane in a liquid buffer, and electric current is applied.
Chemical buffer stabilizes the pH value to the desired value within the gel itself and in the electrophoresis buffer. The choice of buffer also affects the electrophoretic mobility of the buffer counterions and thereby the resolution of the gel. The buffer should also be unreactive and not modify or react with most proteins.
The immobilized pH gradient is obtained by the continuous change in the ratio of immobilines. An immobiline is a weak acid or base defined by its pK value. A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate toward the cathode (negatively charged electrode). As it migrates through a ...
Discontinuity is based on four parameters: gel structure, pH value of the buffer, ionic strength of the buffer, and the nature of the ions in the gel and electrode buffer. The electrode buffer contains glycine. Glycine has very low net charge at pH 6.8 of stacking gel, so it has low mobility.