단백질 분리 및 전기영동

문서 내 토픽

• 1. Lysis buffer

  Lysis buffer는 세포 화합물을 분석을 위해 개방형 세포를 파괴할 때 사용되는 완충액이다. 대부분의 용해 완충액은 용해물의 산도 및 삼투압을 조절하기 위해 염(NaCl)을 함유한다. 때로는 세제(Triton X-100 또는 SDS)를 첨가하여 막 구조를 해체할 수도 있다. 또한 용해 완충액은 동물과 식물의 조직 세포 모두에서 사용할 수 있다.

• 2. Bradford assay

  Bradford assay 단백질 정량(시료 내에 포함된 단백질 양, 농도를 구하는 과정)법 중 하나로 UV-Visible Spectrophotometer를 이용해 흡광도를 측정하고 standard 물질인 BSA를 기준으로 시료에 단백질이 얼마나 들어있는지 측정할 수 있는 방법이다. 산성조건에서는 붉은색이었던 염색약이 푸른색으로 변하게 되고 단백질과 결합을 하게된다. protein-coomassie blue complex수는 단백질의 농도와 비례하며 흡광도와도 비례하게 된다.

• 3. PAGE (폴리아크릴아마이드 젤 전기영동)

  전기영동은 하전된 분자가 전기장에서 반대 전하 쪽으로 이동하는 현상을 이용하여 생체분자를 분리 및 분석하는 방법이다. 단백질 등 대부분의 생체 고분자물질은 전하를 띄고 있어서 전기장에서 이동하는 성질을 가지고 있는데, 이를 바탕으로 젤로 된 지지체를 이용하여 고분자를 전기장 내에서 분리하는 방법을 젤 전기영동이라 한다. 이동 속도는 입자의 전하량, 크기, 모양 등 여러 요인에 의해 결정된다.

• 4. 단백질 농도 측정

  Bradford assay의 방식을 이용하여 standard curve를 구하고, 단백질의 농도를 찾는 실험을 진행하였다. 그 결과 표본의 농도보다 구한 단백질의 농도가 2배 이상 높게 나왔다. BSA의 농도를 정확하게 만드는 것이 Bradford assay 방식을 제대로 이용하기 위해 중요하다는 것을 알게되었다.

• 5. 8% gel과 10% gel 비교

  농도가 다른 8% gel과 10% gel을 이용하여 단백질을 전기영동 했을 때 8% gel에서의 단백질 이동 속도가 더 빠르다는 것을 확인할 수 있었다. 8% gel에서는 gel이 더 많이 얽힌 상태로 있는 10% gel에 비해 작은 크기의 단백질 입자들이 구멍을 통해서 쉽게 빠져나올 수 있기 때문이다.

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• 1. Lysis buffer

  Lysis buffer is a crucial component in various biochemical and molecular biology techniques, as it facilitates the extraction and solubilization of cellular contents, including proteins, nucleic acids, and other biomolecules. The composition of the lysis buffer can vary depending on the specific application, but it typically contains a combination of detergents, salts, buffers, and sometimes reducing agents or protease inhibitors. The choice of lysis buffer is essential in ensuring the efficient and gentle disruption of cells or tissues, while preserving the integrity and functionality of the target biomolecules. The appropriate selection and optimization of the lysis buffer can significantly impact the downstream analysis and applications, such as protein purification, enzyme activity assays, or gene expression studies. Understanding the principles and considerations in the design and use of lysis buffers is crucial for researchers working in various fields of biology and biochemistry.

• 2. Bradford assay

  The Bradford assay is a widely used colorimetric method for the quantitative determination of protein concentration in a sample. It is based on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, which results in a color change that can be measured spectrophotometrically. The Bradford assay is a simple, rapid, and sensitive technique that can be applied to a wide range of protein samples, including purified proteins, cell lysates, and biological fluids. The assay is particularly useful when working with small sample volumes or when the protein concentration is relatively low. However, it is important to note that the Bradford assay can be influenced by the presence of certain compounds, such as detergents or reducing agents, which can interfere with the dye-protein interaction. Careful sample preparation and the use of appropriate standards are crucial for obtaining accurate and reliable protein concentration measurements using the Bradford assay. Overall, the Bradford assay remains a valuable tool in the field of protein analysis and quantification.

• 3. PAGE (폴리아크릴아마이드 젤 전기영동)

  PAGE (Polyacrylamide Gel Electrophoresis) is a widely used analytical technique in molecular biology and biochemistry for the separation and characterization of proteins, nucleic acids, and other biomolecules based on their size and/or charge. The process involves the migration of charged molecules through a polyacrylamide gel matrix under the influence of an electric field. The separation of molecules is achieved due to the sieving effect of the gel, where smaller molecules move faster through the pores compared to larger ones. PAGE can be performed under denaturing conditions (SDS-PAGE) to separate proteins based on their molecular weight, or under native conditions to preserve the native structure and function of the biomolecules. The technique is highly versatile and can be used for various applications, such as protein profiling, enzyme activity analysis, and DNA/RNA fragment separation. The choice of gel concentration, buffer composition, and running conditions can be optimized to achieve the desired resolution and separation of the target biomolecules. Proper sample preparation, gel casting, and electrophoresis conditions are crucial for obtaining reliable and reproducible results using PAGE.

• 4. 단백질 농도 측정

  Accurate measurement of protein concentration is a fundamental requirement in many areas of biochemistry, molecular biology, and biotechnology. There are several methods available for determining protein concentration, each with its own advantages and limitations. The most commonly used techniques include the Bradford assay, the Lowry assay, the bicinchoninic acid (BCA) assay, and UV absorbance at 280 nm. Each method has its own strengths and weaknesses in terms of sensitivity, accuracy, interference from other compounds, and the amount of sample required. The choice of the appropriate protein quantification method depends on factors such as the sample composition, the available equipment, the required sensitivity, and the specific research or application needs. It is important to carefully select and optimize the protein concentration measurement technique, as accurate protein quantification is crucial for downstream applications, such as enzyme kinetics, protein purification, and immunoassays. Additionally, the use of appropriate standards and controls, as well as proper sample preparation, can help ensure the reliability and reproducibility of protein concentration measurements.

• 5. 8% gel과 10% gel 비교

  The choice between an 8% gel and a 10% gel in polyacrylamide gel electrophoresis (PAGE) depends on the specific requirements of the separation and the characteristics of the target proteins or biomolecules. 8% gels are generally used for the separation of larger proteins or protein complexes with higher molecular weights, typically in the range of 50-200 kDa. The lower acrylamide concentration in the 8% gel creates larger pores, allowing for better separation and resolution of higher molecular weight proteins. This is particularly useful when analyzing proteins that may not be well-resolved on a higher percentage gel. On the other hand, 10% gels are more suitable for the separation of smaller to medium-sized proteins, typically in the range of 10-100 kDa. The higher acrylamide concentration in the 10% gel provides a tighter pore structure, which enhances the separation of smaller proteins and allows for better resolution of closely migrating bands. The choice between 8% and 10% gels also depends on the specific research question, the complexity of the protein sample, and the desired level of resolution. In some cases, a gradient gel with a range of acrylamide concentrations may be used to achieve optimal separation across a broader molecular weight range. Ultimately, the selection of the appropriate gel percentage should be guided by the specific requirements of the experiment, the characteristics of the target proteins, and the desired level of resolution and separation. Careful optimization of the PAGE conditions, including the gel percentage, can significantly improve the quality and reliability of the protein analysis.