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    Western Blotting

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    khairul islam nobin
    This document provides an overview of immunoblotting or Western blotting. It describes the technique as a method to identify and locate specific proteins in a sample based on antibody binding. The key steps involve sample preparation, gel electrophoresis to separate proteins by size, protein transfer to a membrane, blocking to prevent non-specific binding, primary antibody probing to detect the target protein, and detection of bound primary antibodies. The process allows determination of a protein's size and expression level for analytical purposes.

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    Western Blotting

    Uploaded by

    khairul islam nobin
    24 views11 pages
    This document provides an overview of immunoblotting or Western blotting. It describes the technique as a method to identify and locate specific proteins in a sample based on antibody binding. The key steps involve sample preparation, gel electrophoresis to separate proteins by size, protein transfer to a membrane, blocking to prevent non-specific binding, primary antibody probing to detect the target protein, and detection of bound primary antibodies. The process allows determination of a protein's size and expression level for analytical purposes.

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    This document provides an overview of immunoblotting or Western blotting. It describes the technique as a method to identify and locate specific proteins in a sample based on antibody binding. The key steps involve sample preparation, gel electrophoresis to separate proteins by size, protein transfer to a membrane, blocking to prevent non-specific binding, primary antibody probing to detect the target protein, and detection of bound primary antibodies. The process allows determination of a protein's size and expression level for analytical purposes.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    24 views11 pages

    Western Blotting

    Uploaded by

    khairul islam nobin
    This document provides an overview of immunoblotting or Western blotting. It describes the technique as a method to identify and locate specific proteins in a sample based on antibody binding. The key steps involve sample preparation, gel electrophoresis to separate proteins by size, protein transfer to a membrane, blocking to prevent non-specific binding, primary antibody probing to detect the target protein, and detection of bound primary antibodies. The process allows determination of a protein's size and expression level for analytical purposes.

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    © All Rights Reserved
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    Immunoblotting

    Nikkon Sarker
    Lecturer, Department of Microbiology

    1
    Western Blotting
    • Analytical technique used to identify and locate specific proteins in a
    sample (containing mixture of proteins) based on their ability to bind
    to specific antibodies
    • Gives information on:
    • Size of protein (with comparison to a size marker or ladder in kDa)
    • Expression amount of protein(with comparison to a control such as
    untreated sample or another cell type or tissue)

    2
    Steps
    • Sample preparation
    • Gel Electrophoresis
    • Transfer
    • Blocking
    • Antibody probing
    • Detection

    3
    Sample Preparation
    • Samples can be taken from whole tissues, cell culture, bacteria,
    viruses, environmental samples etc that are homogenized in a buffer
    to protect the protein of interest from degradation
    • Solid tissues broken down mechanically (eg. by blender)
    • Detergents, salts or buffers may be added to encourage lysis (breaking
    of cell membrane) and solubilize proteins
    • Done at low temperatures to prevent protein denaturation

    4
    Gel Electrophoresis
    • SDS-PAGE (Sodium dodecyl sulfate - Polyachralamide gel
    electrophoresis)
    • Separated by molecular weight

    5
    Transfer
    • Proteins moved from within the gel to a membrane made of
    nitrocellulose or polyvinylidene difluoride (PVDF)
    • Membrane is placed on top of gel, with a stack of filter papers placed
    on top of membrane
    • Entire stack is placed in a buffer solution, which moves up the paper
    through capillary action, bringing the proteins up with it
    • Another method is called electroblotting, where an electric current
    pulls the proteins from the gel to the membrane

    6
    Transfer

    7
    Blocking
    • As the membrane is able to bind protein, steps are taken to prevent
    interactions between the membrane and the antibody used for
    detecting the target protein
    • Blocking agent: Dry milk, Bovine serum albumin

    8
    Antibody probing
    • A primary antibody is added at an appropriate dilution and incubated
    with the membrane. It will bind to the target protein if it is present
    • Membrane rinsed to remove unbound primary antibody. In order to
    detect the antibodies which have bound, a second antibody (or
    “conjugate”) is added. These are anti-immunoglobulin antibodies.

    9
    Detection
    • After excess second antibody is washed off, a substrate is added
    • precipitates upon reaction with the conjugate resulting in a visible
    band where the primary antibody bound to the protein
    • An isotope-labeled primary antibody can also be used, which can be
    detected directly by X-ray film and does not require the secondary
    antibody

    10
    Summary

    11

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