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    Western blotting

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    hulwhulm

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    © All Rights Reserved
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    Western blotting

    Uploaded by

    hulwhulm
    2 views2 pages

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    © © All Rights Reserved

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    Western blotting

    Uploaded by

    hulwhulm

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
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    Western blotting:

    Western blotting is an important technique used in cell and molecular biology. By using a western blot,
    researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells.
    In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through
    gel electrophoresis. After separating the protein mixture, it is transferred to a membrane producing a band
    for each protein. The membrane is then incubated with labels antibodies specific to the protein of interest.
    The unbound antibody is washed off leaving only the bound antibody to the protein of interest. The bound
    antibodies are then detected by developing the film. As the antibodies only bind to the protein of interest,
    only one band should be visible. The thickness of the band corresponds to the amount of protein present;
    thus doing a standard can indicate the amount of protein present.

    Technique:

    1. Sample preparation: Protein can be extracted from different kind of samples, such as tissue or
    cells lysates in a sample buffer containg SDS, glycerol, and loading dye . The sample is heated
    to near- boiling, which denatures the protein and allows the SDS to bind to the protein. SDS
    carries negative charge. Glycerol to make samples sink into wells and the Tris base provides
    appropriate pH. The blue dye to visualize samples as gel is run.

    2. Gel electrophoresis: Western blot uses two different types of agarose gel: stacking and separating
    gel. The higher, stacking gel is slightly acidic (pH 6.8) and has a lower acrylamide concentration
    making a porous gel, which separates protein poorly but allows them to form thin, sharply defined
    bands. The lower gel, called the separating, or resolving gel, is basic (pH 8.8), and has a higher
    polyacrylamide content, making the gel's pores narrower. Protein is thus separated by their size
    more so in this gel, as the smaller proteins to travel more easily, and hence rapidly, than larger
    proteins. Once the marker and samples are loaded, a current is run across the gel. With a
    negative pole on the well of the gel, a positive pole on the opposite end of the gel. Because the
    protein is bounded to negatively charged SDS, it is pulled down through the gel to the positive
    pole. The larger the protein, the slower it moves.

    3. Blotting: After separating the protein mixture, it is transferred to a membrane. The transfer is done
    using an electric field oriented perpendicular to the surface of the gel, causing proteins to move out
    of the gel and onto the membrane. The membrane is placed between the gel surface and the positive
    electrode in a sandwich so that the negatively charged proteins can migrate from the gel to the
    membrane.. The sandwich includes a fiber pad (sponge) at each end, and filter papers to protect the
    gel and blotting membrane.
    4. Washing, blocking and antibody incubation: Blocking prevents antibodies from binding to
    the membrane nonspecifically. Blocking is often made with 5% BSA or nonfat dried milk to
    reduce the background. The membrane is then incubated with primary antibody and detected
    using the label secondary antibody which is detected by the signal it produces corresponding to
    the position of the target protein. This signal is captured on a film which is usually developed in a
    dark room.

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