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Biology LibreTexts

12.2J: Immunoblot Procedures

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  • Immunoblot is a technique for analyzing proteins via antigen-antibody specific reactions.

    Learning Objectives

    • Describe how Western blotting allows individuals to detect specific solubilized proteins from serum or cell or tissue extracts

    Key Points

    • In immunoblot techniques such as Western blot analysis, proteins are separated by electrophoresis and transferred onto nitrocellulose sheets, then are identified by their reaction with labeled antibodies.
    • Electrophoresis uses an electric current to separate proteins based on their size. Big proteins migrate slower and are represented by the highest bands on the blot, while small proteins migrate faster and are indicated by the lowest bands on the blot.
    • Immunoblot assays are usually performed to confirm results obtained by other techniques such as ELISA.

    Key Terms

    • sodium dodecyl sulfate: strong detergent agent used to reduce and unfold native protein.
    • blot: method of transferring protein, DNA, or RNA onto a carrier membrane.

    Immunoblot procedures like protein blotting, or Western blotting, allow individuals to detect specific solubilized proteins from extracts made from cells or tissues, before or after any purification steps.

    Immunoblotting Procedures

    Figure: Immunoblot: proteins separated by molecular weight and represented by a dark band on a blot.

    This analytic technique proceeds in the following steps. Proteins are generally separated by size using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After this, they are transferred to a synthetic membrane via dry, semi-dry, or wet blotting methods. In the electric field generated by a power supply, the proteins coated with negatively charged SDS migrate toward the positive electrode. As the proteins migrate out of the gel, they are captured on a membrane. Protein binding to the membrane is an irreversible mechanism. Membranes can be of the nitrocellulose, polyvinylidene difluoride (PVDF), or nylon variety. The membrane can then be blocked with serum albumin or milk solution to prevent non-specific antibody binding. This is followed by probing with antibodies specific to the protein being studied on the membrane, a method that is similar to immunohistochemistry, but without a need for fixation. This technique exploits the specificity inherent in antigen-antibody recognition. Detection is typically performed using chromogen or peroxide-linked secondary antibodies to catalyze a chromogenic or chemiluminescent reaction.

    Applications of Immunoblotting

    Western blotting is a routine molecular biology method that can be used to semi-quantitatively compare protein levels between extracts. The size separation, prior to blotting, allows the protein molecular weight to be gauged, as compared with known molecular weight markers. Immunoblots are most often used in research settings and are usually performed to confirm results from ELISA or other immunoassays. In clinical diagnostic settings, immunoelectrophoresis is applied, which involves the electrophoresis of serum or urine followed by immunodiffusion. The size and position of precipitation bands provides the same type of information about antibody amount as the double immunodiffusion method.