Western blot 실험 방법

본 내용은 " [예방약학실험2] Western blot " 의 원문 자료에서 일부 인용된 것입니다. 2023.06.23

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• 1. Western blotting

  Western blotting (immunoblotting)은 샘플 내 특정 단백질의 존재 여부, 양, 크기를 측정하는 실험이다. 단백질이 소량이어도 검출 가능하고, 다양한 종류의 단백질이 혼합된 시료를 검사할 때도 항체의 기질 특이성 덕분에 원하는 단백질만 검출할 수 있다는 장점이 있다.

• 2. Western blot 실험 순서

  Western blot 실험은 크게 5개의 순서로 이루어진다. 1) SDS-PAGE를 시행하여 단백질을 크기별로 분리, 2) 분리된 단백질을 membrane에 전이, 3) blocking solution을 첨가하여 비특이적 결합 방지, 4) 타겟 단백질과 결합하는 1차 항체 투여, 5) 결합하지 않은 항체를 씻어내고 발색효소와 결합한 2차 항체 투여하여 밴드 확인.

• 3. 1차 항체와 2차 항체의 필요성

  Western blot에서 1차 항체와 2차 항체 모두 필요한 이유는 1차 항체가 타겟 단백질에 직접 결합하지만 발색을 위해서는 발색효소와 결합한 2차 항체가 필요하기 때문이다. 2차 항체는 1차 항체의 Fc 부분과 결합하고 발색효소와 컨쥬게이션 되어 있어 발색반응을 통해 밴드를 시각화할 수 있다.

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• 1. Western blotting

  Western blotting is a powerful analytical technique used to detect and quantify specific proteins in a complex mixture. It involves the separation of proteins by size using gel electrophoresis, transfer to a membrane, and subsequent detection using specific antibodies. This method is widely used in various fields of biology and medicine, as it allows researchers to study the expression, modification, and interactions of proteins in cells and tissues. The key steps in Western blotting include sample preparation, gel electrophoresis, protein transfer, blocking, primary and secondary antibody incubation, and signal detection. Each step is crucial for obtaining reliable and accurate results. Western blotting is a versatile and sensitive technique that provides valuable insights into the molecular mechanisms underlying biological processes and disease pathogenesis. Its applications range from basic research to clinical diagnostics, making it an indispensable tool in the field of protein analysis.

• 2. Necessity of primary and secondary antibodies in Western blotting

  The use of primary and secondary antibodies in Western blotting is essential for the specific and sensitive detection of target proteins. The primary antibody is a specific antibody that binds directly to the target protein of interest. It is the key component that provides the specificity of the detection, as it recognizes and binds to a unique epitope or region on the target protein. The primary antibody can be raised against a purified protein, a synthetic peptide, or a recombinant protein, and it can be monoclonal or polyclonal in nature. The secondary antibody, on the other hand, is a general antibody that binds to the primary antibody. It is typically conjugated with a reporter molecule, such as an enzyme (e.g., horseradish peroxidase) or a fluorescent dye. The secondary antibody amplifies the signal, as multiple secondary antibodies can bind to a single primary antibody, resulting in a stronger and more detectable signal. The use of primary and secondary antibodies offers several advantages: 1. Specificity: The primary antibody ensures the specific detection of the target protein, even in a complex mixture of proteins. 2. Sensitivity: The signal amplification provided by the secondary antibody allows for the detection of even low-abundance proteins. 3. Flexibility: Different secondary antibodies can be used with the same primary antibody, allowing for the use of various detection methods (e.g., chemiluminescence, fluorescence). 4. Cost-effectiveness: Primary antibodies are often more expensive than secondary antibodies, so the use of a single primary antibody with multiple secondary antibodies can be more cost-effective. In summary, the combination of primary and secondary antibodies in Western blotting is essential for the specific and sensitive detection of target proteins, making it a powerful tool in various fields of biological research and clinical diagnostics.