Electrophoresis Chapter 3-Lecture 7 If a mixture of electrically charged biomolecules is placed in an electric field of field strength E, they will freely move towards the electrode of opposite charge. Dr Gihan Gawish Chapter 3-Lecture 7

Theory of Electrophoresis Electrophoretic separations are based upon the fact that the electrical force (F) on a charged particle (ion) in an electrical field (E) is proportional to the charge of the particle (q), F = qE The migration of the charged particle in the electric field, called the electrophoretic mobility (μ), μ = v/E = q/f Dr Gihan Gawish

Theory of Electrophoresis Move at quite different depending on physical characteristics of the molecule experimental system used. Dr Gihan Gawish

Theory of Electrophoresis Eq = עf f is the frictional coefficient, describes frictional resistance to mobility and depends on a number of factors such as the mass of the molecule ע is the velocity E is the electric field q is the net charge on molecule Dr Gihan Gawish

1- Moving Boundary Electrophoresis Electrophoresis in a free solution. the separation of colloids through electrophoresis the motion of charged particles through a stationary liquid under the influence of an electric field. Developed by Arne Tiselius in 1937. Tiselius was awarded the 1948 Nobel Prize in chemistry for his work Dr Gihan Gawish

Tiselius Electrophoresis Moving boundary electrophoresis is electrophoresis in a free solution. The apparatus includes a U-shaped cell filled with buffer solution and electrodes immersed at its ends. On applying voltage, the compounds will migrate to the anode or cathode depending on their charges. The sample applied could be any mixture of charged components like a serum Dr Gihan Gawish

Tiselius Electrophoresis Moving boundary electrophoresis is electrophoresis in a free solution. The principle is the motion of charged particles through a stationary liquid under the influence of an electric field. 59% 8% 5% 12% 16% Dr Gihan Gawish

2- Zone Electrophoresis separation cellulose acetate electrophoresis. A drop of sample is applied in a band to a thin sheet of supporting material, like paper, that has been soaked in a slightly-alkaline salt solution. Plasma protein example: At pH 8.6, which is commonly used, all the proteins are negatively charged, but some more strongly than others. Dr Gihan Gawish

Zone Electrophoresis A direct current can flow through the paper because of the conductivity of the buffer The serum proteins move toward the positive electrode. The stronger the negative charge on a protein, the faster it migrates. Dr Gihan Gawish

Zone Electrophoresis After 20 min, the current is turned off the proteins stained to make them visible The separated proteins appear as distinct bands. serum albumin moves closest to the positive electrode, why? Dr Gihan Gawish

3- Gel Electrophoresis The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases the gel is a cross linked polymer whose composition and porosity is chosen based on: the specific weight composition of the target to be analyzed. Dr Gihan Gawish

Gel Electrophoresis larger nucleic acids (greater than a few hundred bases) the preferred matrix *agarose. *Acrylamide, in contrast to *Polyacrylamide, is a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning. Dr Gihan Gawish

Gel Electrophoresis- Visualization stained Ethedium bromide Silver coomassie blue UV Radioactivity Photograph Autoradiogram Dr Gihan Gawish

Gel Electrophoresis- Visualization If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Incomplete separation of the components overlapping bands indistinguishable smears multiple unresolved components. Dr Gihan Gawish

Gel Electrophoresis- Visualization The bands observed (unknown molecular weight) can be compared to those of the known (Molecular weight size markers) in order to determine their size. Molecular weight size markers contain a mixture of molecules of known sizes. marker run on one lane in the gel parallel to the unknown samples Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed means they are approximately the same size Dr Gihan Gawish

Applications of Gel Electrophoresis Forensics, Molecular biology, Genetics Microbiology Biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera Animation Dr Gihan Gawish