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    BS-Chemistry 7 Semester-Org (Morning) : Subject

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    Muhammad Asad Faroqui
    The document discusses different electrophoresis techniques used to separate charged particles like proteins and nucleic acids, including paper electrophoresis which uses a paper strip and gel electrophoresis which uses an agarose or polyacrylamide gel as a support matrix. It explains the principles, processes, types like horizontal and vertical gel electrophoresis, and applications of these techniques in fields like forensics, molecular biology and biochemistry.

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    BS-Chemistry 7 Semester-Org (Morning) : Subject

    Uploaded by

    Muhammad Asad Faroqui
    16 views5 pages
    The document discusses different electrophoresis techniques used to separate charged particles like proteins and nucleic acids, including paper electrophoresis which uses a paper strip and gel electrophoresis which uses an agarose or polyacrylamide gel as a support matrix. It explains the principles, processes, types like horizontal and vertical gel electrophoresis, and applications of these techniques in fields like forensics, molecular biology and biochemistry.

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    gel electrophoresis

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    The document discusses different electrophoresis techniques used to separate charged particles like proteins and nucleic acids, including paper electrophoresis which uses a paper strip and gel electrophoresis which uses an agarose or polyacrylamide gel as a support matrix. It explains the principles, processes, types like horizontal and vertical gel electrophoresis, and applications of these techniques in fields like forensics, molecular biology and biochemistry.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    16 views5 pages

    BS-Chemistry 7 Semester-Org (Morning) : Subject

    Uploaded by

    Muhammad Asad Faroqui
    The document discusses different electrophoresis techniques used to separate charged particles like proteins and nucleic acids, including paper electrophoresis which uses a paper strip and gel electrophoresis which uses an agarose or polyacrylamide gel as a support matrix. It explains the principles, processes, types like horizontal and vertical gel electrophoresis, and applications of these techniques in fields like forensics, molecular biology and biochemistry.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 5

    Subject:

    Physical techniques in Biochemistry


    Assignment on:
    Paper and gel electrophoresis

    Submitted to:
    Dr. Sajid Mehmood

    Submitted by:
    Muhammad Asad Faroqui
    Bsf1704526

    BS-Chemistry 7th semester-Org


    (Morning)
    Paper and gel electrophoresis
    Introduction:

    The transport of particles through a solvent by application of an electric field is called as


    electrophoresis. Most of the polymers (containing macromolecules) are electrically charged and
    will therefore move in an electric field. Electrophoresis is useful in identification and structure
    determination of such big molecules.

    Definition:

    • Electro means Electricity


    • Phoresis means Separation
    • Electrophoresis may be defined as the migration of the charged particle through a solution
    under the influence of an external electrical field.

    Key points:

    • This technique is used to determine protein molecular weights, to distinguish molecules by


    virtue of their net charge or their shape and to separate different molecular species
    quantitatively.
    • Rate of movement of macromolecules in an electric field is useful parameter to know any
    change in amino acid regarding its charge.
    • Electrophoresis is similar to chromatography. Electric field is used as a dragging force.
    • Technique is simple, very effective and clean.
    • Large number of samples can be separated, identified and quantitatively measured.

    Paper electrophoresis:

    ➢ Paper electrophoresis (PE) is useful for the separation of small-charged molecules, such as
    amino acids and small proteins using a strip of paper (chromatography paper).
    ➢ In this technique, the motion of colloidal particle of solution occurs leading to subsequent
    separation along the paper strip.
    ➢ PE is easier in comparison to gel electrophoresis.
    ➢ It does not require matrix preparation and it does not contain charges that interfere with the
    separation of compounds.

    Process:

    ➢ A strip of filter paper is moistened with buffer and the ends of the strip are immersed into
    buffer reservoirs containing the electrodes.
    ➢ The samples are spotted in the center of the paper and high voltage is applied.
    ➢ Application of high voltage causes less diffusion of small molecules giving better
    resolution and it take less time to complete the process.
    ➢ Spots migrate according to their charges.
    ➢ After electrophoresis, separated components can be detected by variety of staining
    techniques, depending upon their chemical composition.

    Applications of Paper Electrophoresis

    ➢ A simple, inexpensive, and accurate laboratory procedure for various research and clinical
    studies.
    ➢ Easily available and easy to handle, allowing new methodologies to be tried and developed
    with convenience.
    ➢ Clinical applications of PE include study of sickle cell disease, hemoglobin abnormalities,
    and separation of blood clotting factors and serum plasma proteins from blood sample.
    ➢ Used in separation and identification of alkaloids.
    ➢ Used for testing suitability of municipal water supplies, toxicity of water, and other
    environmental components.
    ➢ Drug-testing industry uses paper electrophoresis to determine presence of illegal or
    recreational drugs in job applicants and crime suspects. Since 1950s used by the
    investigators and in forensics to analyze inks used in currency to check the counterfeiters.
    Gel electrophoresis:

    ➢ It makes the use of gel as a support matrix.


    ➢ Most popular and commonly used method used for both analytical and preparative
    processes.
    ➢ It is the most common method to carry out the process of electrophoresis.

    Principle:

    In this porous gel matrix is used which consist of the cross-linked polymer network.
    Through this network, molecules of different size, charge and shape pass through. This relies upon
    the fact that negatively charged molecule will attract towards the positive end and vice versa. After
    the migration, bands will appear on the gel matrix at different levels those which lag behind will
    be the heavy molecules and those which moves faster are lighter molecules through the pores of
    the gel matrix.

    Types of Gel electrophoresis

    1. Horizontal
    2. Vertical

    Agarose gel electrophoresis:

    It is a type of horizontal electrophoresis. To carry out this kind of electrophoresis, following


    steps involve:
    ➢ First, take agarose into the water to make the slurry or to dissolve the agarose. Cool the
    agarose solution, and then transfer it to the casting tray containing comb.
    ➢ Add electrophoresis buffer (Tris-acetate EDTA buffer) to cover the gel up to 1mm.
    ➢ Then, take the DNA sample.
    ➢ Add the desired amount of 6X gel loading buffer.
    ➢ After that, take out the comb and slowly load the sample mixture containing tracking dye
    (Bromophenol blue) into the wells through the micropipette.
    ➢ Close the lid of the gel tank, and turn on the power supply so that DNA can migrate towards
    the positive electrode.
    ➢ Run the gel, until the bromophenol blue migrates on an appropriate distance through a
    gel.
    ➢ At last, remove the gel tray and place is in a UV Trans illuminator, to see the orange-red
    colored DNA bands.

    Applications of Gel electrophoresis:

    ➢ Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and


    biochemistry.
    ➢ Analysis of lipoproteins.
    ➢ Gel electrophoresis is used for the isolation of nucleic acid especially DNA.

    ➢ Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting.

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