Protein Electro-transfer and Western blot

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1. Western blotting

Western blotting은 단백질에 대한 blotting 기술을 뜻하며, 항원-항체 반응을 이용한다. 단백질 혼합물을 polyacrylamide gel에서 SDS-PAGE를 이용하여 크기에 따라 분리하고 membrane으로 electro-transfer하여 흡착시킨 후 membrane 표면에서 항체를 이용하여 원하는 단백질의 존재를 확인하는 전반적인 과정을 의미한다.
2. SDS의 역할 및 원리

SDS-PAGE는 SDS가 단백질에 일정 간격으로 결합하여 charge density를 일정하게 하고, 단백질을 변형시켜 모든 단백질이 일정 모양(선형)을 갖게 함으로써 단백질이 전기장에서 크기(분자량)에만 의존적으로 분리되게 한다.
3. Nitrocellulose membrane으로의 Electro-Transfer

Electro-Transfer는 SDS-PAGE로 분리된 단백질을 membrane(nitrocellulose or PVDF)으로 옮기는 과정이다. 단백질은 모두 음전하를 띠고 있기 때문에 양극으로 이동하면서 gel에 있던 단백질이 그대로 빠져나와 membrane으로 옮겨가게 된다.
4. Blocking and Antibody reaction

Membrane 표면에 항체가 비특이적으로 붙는 것을 방지하기 위해 비특이적인 단백질(Skim milk, bovine serum albumin)로 mebrane 전체를 코팅한다. 그 후 특정 단백질에 대한 1차 항체를 membrane과 반응시키고, 여분의 1차 항체를 wash한 후에 효소가 붙어 있는 2차 항체를 반응시킨다.
5. Detection

항체에 효소 부착 2차 항체를 반응한 뒤 효소의 기질 용액을 처리하면 특이적으로 결합한 단백질의 위치에 색 또는 형광이 나타나는 것을 볼 수 있는데, 이를 통해 단백질의 크기와 양을 계산할 수 있다. 이때 사용하는 효소에 대한 기질로는 발색(colorimetic), 형광(Fluorescence), 또는 발광(Luminescence)이 있다.
6. 실험 결과 분석

이번 실험에서 검출하고자 한 단백질인 Naa30 protein은 40kDa의 사이즈를 가지지만, 실험 결과에서 검출된 sample의 protein band들은 모두 65kDa 부근에서 intensity 비율이 높게 나타나는 것으로 보아 다른 크기를 갖고 있는 단백질의 band가 검출된 것을 알 수 있다. 또한, sample 30-1, 30-2, 100-1, 100-2에서는 특히 다양한 band가 존재하는 것을 관찰할 수 있다.
7. Transfer membrane 종류 및 원리

Transfer membrane의 종류는 Nitrocellulose, PVDF, Nylon 등이 있으며, 각각 Electrostatic&Hydrophobic, Convalent&Hydrophobic, Ionic, hydrophobic, and electrostatic의 흡착원리를 이용한다. Nitrocellulose membrane은 한 번에 많은 antibody의 흡착이 가능하지만 재질이 약하고 단백질 결합력이 상대적으로 약하다는 단점이 있다.
8. Transfer buffer 조성

Transfer buffer에 들어있는 Tris-base와 glycine은 전기가 흐를 수 있게 하기 위한 전해질 용액이며, 20% methanol은 단백질이 gel에서 잘 빠져나올 수 있도록 도와주는 역할을 한다.
9. BSA와 Skim milk의 차이

Skim Milk의 경우 blocking 효과는 뛰어나나, 일부 antibody의 antigen과의 recognition site를 masking함으로써 antibody의 antigen 인식을 방해하며, band의 specificity를 떨어뜨리는 경우가 발생한다. BSA의 경우, skim milk에 비해 blocking효과는 떨어지나, 이러한 문제는 나타나지 않는다.
10. Antibody의 정의 및 용도

Antibody(항체)란 antigen(항원)과 결합하는 단백질을 말하며, 혈액과 체액에 녹아있는 Immunoglobulin(Ig)를 뜻한다. Antibody를 이용한 실험들로는 Western blotting 외에도, 면역조직화학, ELISA, 면역형광 세포 염색법, 면역침강법 등이 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 of proteins extracted from cells or tissues. It involves several key steps, including sample preparation, gel electrophoresis, protein transfer to a membrane, antibody incubation, and signal detection. This technique is widely used in various fields of biology and medicine to study protein expression, post-translational modifications, and protein-protein interactions. The ability to separate and identify specific proteins from a complex mixture makes Western blotting an indispensable tool for researchers in understanding cellular processes, disease mechanisms, and developing targeted therapies. The technique requires careful optimization of each step to ensure accurate and reliable results, and the interpretation of Western blot data requires a deep understanding of the underlying principles and potential pitfalls. Overall, Western blotting remains a crucial technique in the arsenal of modern molecular biology and biochemistry research.
2. SDS의 역할 및 원리

SDS (Sodium Dodecyl Sulfate) plays a crucial role in the Western blotting process by denaturing and solubilizing proteins. The primary function of SDS is to disrupt the non-covalent interactions within and between proteins, causing them to unfold and adopt a linear, rod-like structure. This unfolding ensures that the proteins are separated based on their molecular weight during the gel electrophoresis step, rather than their native three-dimensional structure. The negatively charged SDS molecules also bind to the denatured proteins in a roughly constant mass-to-charge ratio, allowing the proteins to migrate through the gel based on their molecular weight alone. The use of SDS is essential for achieving consistent and reproducible separation of proteins, which is a fundamental requirement for accurate Western blot analysis. Additionally, the denaturing and solubilizing properties of SDS help to prevent protein aggregation and ensure that the target proteins are accessible for antibody binding during the detection step. Understanding the underlying principles of SDS and its role in the Western blotting process is crucial for optimizing the technique and interpreting the results accurately.
3. Nitrocellulose membrane으로의 Electro-Transfer

The transfer of proteins from the gel to a nitrocellulose membrane during the Western blotting process is a critical step that ensures the proteins are accessible for antibody binding and detection. The electro-transfer process involves applying an electric current to the gel-membrane sandwich, which causes the denatured proteins to migrate from the gel and become immobilized on the nitrocellulose membrane. The choice of nitrocellulose as the transfer membrane is due to its high protein-binding capacity, which allows for efficient and uniform capture of the separated proteins. The electro-transfer step must be carefully optimized in terms of voltage, current, and duration to ensure complete and even transfer of the proteins, without causing distortion or loss of resolution. Factors such as buffer composition, temperature, and the presence of methanol can also influence the efficiency and quality of the protein transfer. Understanding the principles of electro-transfer and the properties of nitrocellulose membranes is essential for obtaining reliable and reproducible Western blot results.
4. Blocking and Antibody reaction

Blocking and antibody incubation are crucial steps in the Western blotting process, as they ensure the specific and efficient detection of the target proteins. The blocking step involves incubating the nitrocellulose membrane with a solution containing a non-specific protein, such as bovine serum albumin (BSA) or non-fat dry milk, to prevent non-specific binding of the primary and secondary antibodies. This is essential to minimize background signals and improve the signal-to-noise ratio, allowing for accurate quantification of the target proteins. The subsequent incubation with the primary antibody, which is specific to the target protein, allows for the formation of antigen-antibody complexes on the membrane. The choice of primary antibody, its concentration, and the incubation conditions (time, temperature, and buffer composition) must be carefully optimized to ensure optimal binding and specificity. The final step involves the incubation with a secondary antibody, which is conjugated with a reporter molecule (e.g., enzyme or fluorescent dye) and binds to the primary antibody. This enables the detection and visualization of the target proteins on the membrane. Understanding the principles of blocking, antibody selection, and incubation conditions is essential for obtaining reliable and specific Western blot results.
5. Detection

The detection step in Western blotting is the final and crucial stage that allows for the visualization and quantification of the target proteins. This step involves the use of a reporter molecule, such as an enzyme or a fluorescent dye, which is conjugated to the secondary antibody. When the secondary antibody binds to the primary antibody-antigen complex, the reporter molecule is activated, generating a detectable signal. The most common detection methods include chemiluminescence, where the enzyme-catalyzed reaction produces light, and fluorescence, where the fluorescent dye emits light upon excitation. The choice of detection method depends on factors such as the sensitivity required, the availability of equipment, and the nature of the target proteins. Careful optimization of the detection conditions, including the concentration of the secondary antibody, the incubation time, and the substrate or excitation parameters, is essential to ensure a linear and quantitative relationship between the target protein amount and the signal intensity. The interpretation of the detection results, including the analysis of band intensity and the comparison of samples, requires a thorough understanding of the underlying principles and potential sources of error. Proper detection and data analysis are crucial for drawing accurate conclusions from Western blot experiments.
6. 실험 결과 분석

The analysis of Western blot results is a critical step in the overall process, as it allows researchers to draw meaningful conclusions from the experimental data. This analysis involves several key aspects, including the evaluation of band intensity, the comparison of protein expression levels across different samples, and the assessment of potential post-translational modifications or protein interactions. The interpretation of Western blot results requires a deep understanding of the underlying biology, the experimental design, and the potential sources of variability and error. Factors such as sample preparation, protein loading, transfer efficiency, and antibody specificity can all influence the observed band patterns and intensities. Careful normalization of the data, using appropriate loading controls or reference proteins, is essential to ensure accurate quantification and comparison of protein levels. Additionally, the analysis should consider the potential limitations of the Western blot technique, such as the inability to detect low-abundance proteins or the potential for cross-reactivity of antibodies. By combining the technical expertise in Western blotting with a thorough understanding of the biological context, researchers can effectively analyze and interpret the experimental results, leading to meaningful insights and informed decision-making.
7. Transfer membrane 종류 및 원리

The choice of transfer membrane is a critical factor in the success of the Western blotting process, as it directly impacts the efficiency and quality of protein transfer from the gel to the membrane. The two most commonly used transfer membranes are nitrocellulose and polyvinylidene difluoride (PVDF). Nitrocellulose membranes have a high protein-binding capacity and are widely used due to their ease of handling and compatibility with various detection methods. PVDF membranes, on the other hand, offer higher mechanical strength and chemical resistance, making them more suitable for certain applications, such as the detection of low-abundance proteins or the use of harsh buffers. The underlying principle of protein transfer to these membranes is the same, involving the application of an electric current that drives the denatured proteins from the gel to the membrane. However, the specific properties of each membrane, such as pore size, surface charge, and hydrophobicity, can influence the efficiency and quality of the transfer, as well as the subsequent antibody binding and detection. Understanding the characteristics and advantages of different transfer membranes is crucial for optimizing the Western blotting protocol and obtaining reliable and reproducible results.
8. Transfer buffer 조성

The composition of the transfer buffer used in the Western blotting process is a critical factor that can significantly impact the efficiency and quality of protein transfer from the gel to the membrane. The transfer buffer typically contains a combination of the following components: 1. Tris and glycine: These provide the necessary ionic strength and pH for the electrophoretic transfer of proteins. 2. Methanol: Methanol is often included in the buffer to help denature and solubilize the proteins, as well as to improve the binding of proteins to the membrane. 3. SDS: The inclusion of a small amount of SDS can help maintain the denatured state of the proteins and improve their transfer. 4. Other additives: Depending on the specific application, the buffer may also contain other components, such as EDTA or reducing agents, to further enhance the transfer process. The optimal composition and concentration of these buffer components must be carefully determined through empirical testing, as they can vary depending on the specific proteins of interest, the type of membrane used, and the experimental conditions. Factors such as the buffer pH, ionic strength, and the presence of organic solvents can all influence the efficiency and uniformity of the protein transfer. Understanding the principles behind the transfer buffer composition and its optimization is crucial for obtaining high-quality and reproducible Western blot results.
9. BSA와 Skim milk의 차이

Bovine serum albumin (BSA) and skim milk are both commonly used as blocking agents in the Western blotting process, but they have distinct differences in their composition and properties. BSA is a purified protein derived from bovine serum, and it is a widely used blocking agent due to its high purity and lack of interfering components. BSA is a relatively inert protein that effectively blocks non-specific binding sites on the membrane, allowing for more specific antibody-antigen interactions during the detection step. BSA is often preferred when the target protein is known to have low abundance or when the primary antibody is raised in the same species as the BSA (e.g., a mouse primary antibody and BSA from bovine serum). In contrast, skim milk is a complex mixture of proteins, carbohydrates, and other components derived from non-fat milk. While skim milk is also an effective blocking agent, it may contain antibodies or other proteins that can potentially cross-react with the target protein or the primary antibody, leading to increased background signal or non-specific binding. Skim milk is often used as a more cost-effective alternative to BSA, and it may be preferred when the target protein is highly abundant or when the primary antibody is raised in a different species than the BSA. The choice between BSA and skim milk as a blocking agent depends on the specific requirements of the Western blotting experiment, the characteristics of the target protein, and the properties of the primary antibody. Careful evaluation and optimization of the blocking agent can significantly improve the specificity and sensitivity of the Western blot analysis.
10. Antibody의 정의 및 용도

Antibodies are Y-shaped glycoproteins produced by the immune system in response to the presence of foreign substances, known as antigens. They play a crucial role in the Western blotting technique, as they are used to specifically detect and quantify target proteins. The primary function of antibodies in Western blotting is to bind to the target protein with high affinity and specificity, allowing for its identification and quantification. Antibodies are classified into different types, such as polyclonal and monoclonal, based on their origin and specificity. Polyclonal antibodies are produced by a mixture of B cells and recognize multiple epitopes on the target protein, while monoclonal antibodies are derived from a single B cell clone and bind to a specific epitope. The versatility of antibodies in Western blotting lies in their ability to be used for various applications, including: 1. Protein detection and quantification: Antibodies are used to detect the presence and measure the abundance of target proteins in complex mixtures. 2. Protein localization: Antibodies can be used to determine the subcellular localization of proteins within cells or tissues. 3. Protein-protein interactions: Antibodies can be used to identify and study the interactions between different proteins. 4. Post-translational modifications: Antibodies can be used to detect and analyze specific post-translational modifications, such as phosphorylation, acetylation, or glycosylation, on target proteins. The careful selection and optimization of antibodies, including their specificity, affinity, and concentration, are crucial for obtaining reliable and accurate Western blot results. Understanding the definition and diverse applications of antibodies is essential for effectively utilizing this powerful tool in the field of molecular biology and biochemistry.

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Protein electro-transfer and Western blot 실험보고서

1. Western blot Western blot은 찾고자 하는 protein의 antigen epitope와 반응하는 antibody를 이용하여 단백질 혼합물 중에서 원하는 단백질(antigen)을 찾아내는 방법이다. 전기 영동을 이용하여 단백질을 gel 상에서 크기에 따라 분리한 후, membrane에 transfer한다. Membrane에 발광하는 ...

2025.01.16 · 의학/약학

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