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Western Blotting 레포트

EGFR과 HA tag를 포함한 pBabe plasmid vector를 HEK-293T cell에 transfection시킨 후, lysis하여 lysate을 생성하고, transfection이 잘 되어서 EGFR gene이 발현되었는지, 또 만들어진 Protein의 양은 얼마나 되는지 Western blotting을 통해 확인한 실험에 대한 레포트입니다. 레포트에 시간을 많이 들여 A+ 받았던 과목이기 때문에, 도움이 되실 거라 생각합니다.
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최초등록일 2024.08.29 최종저작일 2023.11
분자생물학 실험레포트 실험보고서 웨스턴블랏
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Western Blotting 레포트
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    이 내용은 AI를 통해 자동 생성된 정보로, 참고용으로만 활용해 주세요.
    • 전문성
    • 명확성
    • 실용성
    • 유사도 지수
      참고용 안전
    • 🔬 실험 과정의 상세하고 체계적인 프로토콜 제공
    • 📊 Western Blotting 기법의 심도 있는 이론적 배경 설명
    • 🧬 단백질 분석의 전문적이고 실용적인 가이드라인 제시

    미리보기

    소개

    EGFR과 HA tag를 포함한 pBabe plasmid vector를 HEK-293T cell에 transfection시킨 후, lysis하여 lysate을 생성하고, transfection이 잘 되어서 EGFR gene이 발현되었는지, 또 만들어진 Protein의 양은 얼마나 되는지 Western blotting을 통해 확인한 실험에 대한 레포트입니다.
    레포트에 시간을 많이 들여 A+ 받았던 과목이기 때문에, 도움이 되실 거라 생각합니다.

    목차

    1. Background
    1-1 Western Blotting
    1-2 Sample preparation
    1-3 Gel preparation 및 Electrophoresis
    1-4 SDS-PAGE
    1-5 Transfer
    1-6 Buffer (Sample loading buffer, Running buffer, Transfer buffer)
    1-7 Blocking
    1-8 Abs reaction
    1-9 Detection

    2. Purpose and Method ------------------------
    2-1 Purpose
    2-2 Method

    3. Result -------------------------------------
    3-1 Western blotting band picture
    3-2 Conclusion and Discussion

    4. Reference ----------------------------------

    본문내용

    1-1 Western Blotting
    1) 정의
    - 단백질을 감지하고, 양을 확인하여 분자량을 측정할 수 있는 방법
    - 찾고자 하는 단백질의 antigen epitope과 반응하는 antibody를 이용하여 단백질 혼합물 중에서 원하는 단백질(antigen)만을 찾아내는 방법
    - 단백질을 polyacrylamide gel에서 electrophoresis를 이용하여 분리하고, nitrocellulose membrane에 옮긴 후, 찾고자 하는 단백질의 primary antibody와 primary antibody와 반응하는 secondary antibody를 이용하여 band를 형상화하는 과정
    2) 특징
    - Protein sample을 얻는 Sample preparation, Electrophoresis를 위한 Gel preparation, Transfer, Antibody reaction 등의 여러 개별 단계가 포함된다.
    - Southern Blot이나 Northern Blot 과정에는 대개 Agarose gel electrophoresis를 포함 하는 반면, Western Blot은 SDS-PAGE(Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)를 포함한다.
    - 여러 단백질 중에서 원하는 단백질만을 시각화하여 검출하는 방법으로 fluorescence (fluorescein isothiocyanate), enzyme reaction (peroxidase, alkaline phosphatase, glucose oxidase), radioactivity 등의 방법이 이용되고 있다.

    참고자료

    · Western Blot, 분자세포생물학백과
    · https://terms.naver.com/entry.naver?docId=5569289&cid=61233&categoryId=61233
    · Western Blot, 다인바이오주식회사
    · http://www.dynebio.co.kr/yc/bbs/board.php?bo_table=data1&wr_id=14&sfl=wr_subject&stx=west&sop=and
    · Cell lysis, 생명과학대사전
    · https://terms.naver.com/entry.naver?docId=1612025&cid=50317&categoryId=50317
    · Bradford assay, BIO-RAD
    · https://www.bio-rad.com/ko-kr/applications-technologies/western-blotting-sample-preparation-techniques?ID=PQEEHGSCUMB7
    · SDS-PAGE, 분자세포생물학백과
    · https://terms.naver.com/entry.naver?docId=5751411&cid=61233&categoryId=61233
    · Transfer buffer, BIO-RAD
    · https://www.bio-rad.com/ko-kr/applications-technologies/western-blotting-transfer-techniques?ID=PQEEOP70KWE7
    · Trans blot, BIO-RAD
    · Trans-Blot Turbo Transfer System – Bio-Rad

  • AI와 토픽 톺아보기

    • 1. Western Blotting
      Western blotting is a widely used analytical technique in molecular biology and biochemistry that allows for the detection and quantification of specific proteins within a complex mixture. It is a powerful tool for understanding protein expression, post-translational modifications, and interactions. The process involves several key steps, each of which requires careful optimization to ensure accurate and reliable results. Sample preparation is crucial, as it ensures the proteins are in a suitable state for analysis. Gel preparation, including the choice of gel type and composition, can significantly impact the separation and resolution of proteins. SDS-PAGE, the electrophoretic separation of proteins based on their molecular weight, is a fundamental step in the western blotting workflow. The transfer of separated proteins from the gel to a membrane, such as nitrocellulose or PVDF, is another critical step that must be optimized to ensure efficient and uniform protein transfer. The choice of buffers, blocking agents, and antibodies used in the process can also greatly influence the sensitivity and specificity of the assay. Overall, western blotting is a complex and multifaceted technique that requires a deep understanding of the underlying principles and careful attention to detail at each stage to obtain meaningful and reliable results.
    • 2. Sample preparation
      Sample preparation is a crucial step in the western blotting process, as it ensures that the proteins of interest are in a suitable state for analysis. Proper sample preparation involves several key considerations, such as cell lysis, protein extraction, and quantification. The choice of lysis buffer and the inclusion of protease and phosphatase inhibitors can significantly impact the integrity and solubility of the proteins. Protein quantification, often performed using methods like the Bradford or BCA assay, is essential to ensure equal loading of samples onto the gel, which is crucial for accurate comparative analysis. Additionally, the denaturation and reduction of proteins, typically achieved through the use of heat, reducing agents, and SDS, are important steps to ensure the proteins are in a linear conformation and can be effectively separated by SDS-PAGE. Overall, careful attention to sample preparation can greatly improve the reliability and reproducibility of western blotting results.
    • 3. Gel preparation
      Gel preparation is a critical step in the western blotting process, as the choice of gel type and composition can significantly impact the separation and resolution of proteins. The most commonly used gel type for western blotting is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins based on their molecular weight. The concentration of acrylamide in the gel can be varied to optimize the separation of proteins within a specific molecular weight range. For example, higher acrylamide concentrations (e.g., 12-15%) are typically used for the separation of smaller proteins, while lower concentrations (e.g., 8-10%) are better suited for larger proteins. The inclusion of crosslinking agents, such as bis-acrylamide, and the use of appropriate buffers and pH can also influence the pore size and overall gel structure, affecting the resolution and efficiency of protein separation. Additionally, the casting and polymerization of the gel can impact its integrity and consistency, which is crucial for obtaining reliable and reproducible results. Careful optimization of gel preparation parameters is essential for successful western blotting.
    • 4. SDS-PAGE
      SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) is a fundamental step in the western blotting process, as it separates proteins based on their molecular weight. This technique involves the denaturation of proteins using the anionic detergent SDS, which binds to the proteins and confers a uniform negative charge. The denatured proteins are then loaded onto a polyacrylamide gel and subjected to an electric field, causing them to migrate through the gel matrix at a rate inversely proportional to their molecular weight. The choice of gel concentration, buffer composition, and running conditions can significantly impact the separation and resolution of proteins. Proper optimization of SDS-PAGE parameters, such as acrylamide concentration, voltage, and run time, is crucial to ensure the efficient and accurate separation of the target proteins. Additionally, the inclusion of molecular weight markers can aid in the identification and quantification of the proteins of interest. SDS-PAGE is a powerful tool that provides the foundation for the subsequent western blotting analysis.
    • 5. Transfer
      The transfer step in western blotting is essential for the successful detection and analysis of proteins. After the proteins have been separated by SDS-PAGE, they need to be transferred from the gel to a solid support, such as a nitrocellulose or PVDF membrane, to allow for subsequent antibody-based detection. The choice of transfer method, buffer composition, and transfer conditions can significantly impact the efficiency and uniformity of the protein transfer. Commonly used transfer methods include wet, semi-dry, and tank transfer, each with its own advantages and considerations. The selection of the appropriate membrane material and pore size can also influence the binding and retention of the transferred proteins. Optimizing parameters like transfer voltage, current, time, and temperature is crucial to ensure complete and even transfer of the proteins, which is essential for accurate quantification and comparison of protein levels. Proper transfer optimization is a critical step in the western blotting workflow, as it directly impacts the sensitivity and reliability of the final results.
    • 6. Buffer
      Buffers play a crucial role in the western blotting process, as they are used in various stages to maintain the appropriate pH, ionic strength, and other conditions necessary for the successful separation, transfer, and detection of proteins. The choice of buffer composition can significantly impact the efficiency and specificity of each step. For example, the running buffer used in SDS-PAGE must be able to maintain the denatured state of the proteins and facilitate their migration through the gel matrix. The transfer buffer, on the other hand, must support the efficient and uniform transfer of proteins from the gel to the membrane. Additionally, the blocking buffer and antibody diluent buffers used in the immunodetection step can influence the background signal and the specificity of the antibody-antigen interactions. The inclusion of detergents, such as Tween-20, in the washing and antibody incubation buffers can help reduce non-specific binding and improve the signal-to-noise ratio. Careful selection and optimization of buffer compositions at each stage of the western blotting workflow are essential for obtaining reliable and reproducible results.
    • 7. Blocking
      Blocking is a crucial step in the western blotting process, as it helps to reduce non-specific binding of antibodies to the membrane, which can lead to high background signals and decreased sensitivity. The choice of blocking agent and the blocking conditions can significantly impact the success of the western blot. Common blocking agents include non-fat dry milk, bovine serum albumin (BSA), and various synthetic blocking reagents. The concentration and duration of the blocking step must be optimized to effectively cover the available binding sites on the membrane without interfering with the specific antibody-antigen interactions. Factors such as the type of membrane, the target protein, and the antibodies used can all influence the optimal blocking conditions. Additionally, the composition of the blocking buffer, including the presence of detergents and other additives, can affect the blocking efficiency and the overall signal-to-noise ratio. Proper blocking is essential for achieving high-quality, specific, and sensitive western blot results.
    • 8. Antibody reaction
      The antibody reaction is a critical step in the western blotting process, as it allows for the specific detection and quantification of the target proteins. The choice of primary and secondary antibodies, as well as the incubation conditions, can significantly impact the sensitivity, specificity, and reliability of the western blot results. Factors to consider include the antibody specificity, affinity, and compatibility with the target protein and the experimental conditions. The concentration and dilution of the antibodies, as well as the incubation time and temperature, must be optimized to ensure efficient and specific binding to the target proteins. Additionally, the inclusion of blocking agents and detergents in the antibody diluent can help to reduce non-specific interactions and improve the signal-to-noise ratio. Proper antibody selection and optimization of the antibody reaction conditions are essential for obtaining accurate and reproducible western blot data.
    • 9. Detection
      The detection step in western blotting is the final and crucial stage that allows for the visualization and quantification of the target proteins. There are several detection methods available, each with its own advantages and considerations. Commonly used detection methods include chemiluminescence, fluorescence, and colorimetric detection. Chemiluminescence detection, which relies on the enzymatic conversion of a substrate to produce light, is a widely used and highly sensitive method. Fluorescence detection, which utilizes fluorescently labeled secondary antibodies or dyes, offers the advantage of multiplexing and quantitative analysis. Colorimetric detection, such as the use of chromogenic substrates, provides a more straightforward and cost-effective approach, but may have lower sensitivity compared to other methods. The choice of detection method, as well as the optimization of parameters like exposure time, image acquisition, and data analysis, can significantly impact the quality, sensitivity, and reliability of the western blot results. Careful consideration and optimization of the detection step are essential for obtaining meaningful and reproducible data from western blotting experiments.
    • 10. Buffer
      Buffers play a crucial role in the western blotting process, as they are used in various stages to maintain the appropriate pH, ionic strength, and other conditions necessary for the successful separation, transfer, and detection of proteins. The choice of buffer composition can significantly impact the efficiency and specificity of each step. For example, the running buffer used in SDS-PAGE must be able to maintain the denatured state of the proteins and facilitate their migration through the gel matrix. The transfer buffer, on the other hand, must support the efficient and uniform transfer of proteins from the gel to the membrane. Additionally, the blocking buffer and antibody diluent buffers used in the immunodetection step can influence the background signal and the specificity of the antibody-antigen interactions. The inclusion of detergents, such as Tween-20, in the washing and antibody incubation buffers can help reduce non-specific binding and improve the signal-to-noise ratio. Careful selection and optimization of buffer compositions at each stage of the western blotting workflow are essential for obtaining reliable and reproducible results.

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      Western Blotting 기술을 활용하여 EGFR 단백질 발현을 확인하고자 하였으며, 실험 과정에서 발생한 문제점을 해결하고자 노력하였습니다.

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