이화여대 생명과학실험 A+ 리포트(DEAE sepharose anion exchange chromatography)

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1. Column chromatography

크로마토그래피는 주어진 시료 중 정량 분석하고자 하는 성분을 분리하고 확인하는 수단으로, 여러 다른 화합물들이 상이한 두 가지 phases 간에 분포되어 있거나 어느 물질 내의 분리 가능한 부위들 사이에 분포되어 있을 때 그 분포된 정도가 화합물들끼리 서로 다르다는 사실에 기초한 방법이다. 한 가지 상은 정지상(stationary phase)이고 다른 상은 이동상(mobile phase)이다. 이동상은 정지상을 일고 있는 물질 위로 흐르는데, 분리될 시료는 이 이동상을 따라 운반된다. 시료 중의 여러 성분이 이동상과 고정상을 흐르면서 흡착, 분배, 이온 교환 또는 분자 크기 등의 차이로 분리되는 것을 말한다.
2. Adsorption chromatography and partition chromatography

크로마토그래피의 분리메커니즘으로 흡착이론을 이용한 것인데, 사용되는 흡착제로는 활성알루미나 또는 산화마그네슘 등이 있다. 흡착크로마토그래피는 흡착이론을 사용하는데, 흡착이론은 이동상 내에 있는 분석체들이 정지상에 흡착되는 정도의 차이로 분리하는 것이다. 분배 크로마토그래피는 분배이론을 사용하는데, 분배는 분석체들의 정지상과 상호 분배계수의 차이에 의해 분리되는 것이다.
3. Ion exchange chromatography

이온교환 크로마토그래피는 이온 또는 극성 분자들을 고정상에 결합된 음이온 또는 양이온과의 정전기적 인력(electrostatic interaction)을 이용하여 분리하는 방법이다. 음이온(예를 들어, -SO3-) 또는 양이온(예를 들어, -N(CH3)3+)들이 고정상(주로 레진)에 공유결합으로 부착되고, 용질 내 분자들 중 고정상과 반대의 전하를 띠는 것들이 정전기적 인력을 통하여 고정상과 서로 결합하게 된다.
4. Alkaline phosphatase (AP)

Alkaline Phosphatase (AP)는 염기성 환경에서 활성이 높은 효소로 다양한 분자에서 phosphate group을 떼어내는 역할을 한다. AP는 2개의 동일한 polypetide로 이뤄진 homodimer이며 크기는 160~180kda 정도이고 net charge는 (-)이다. AP는 water 또는 lipid solution에서 용해되며, 75~85 ℃에서도 활성을 가진다.
5. Purification of alkaline phosphatase

AP 정제를 위해 E.Coli에서 AP를 over expression시키고, osmotic shock을 통해 outer membrane을 파괴하여 intermembrane space에 존재하는 AP를 분리한다. 원심분리를 통해 AP가 포함된 supernatant를 얻고, 이를 dialysis하여 AP를 정제한다. 이후 heat shock을 통해 다른 단백질들을 불활성화시키고, DEAE sepharose anion exchange chromatography를 이용하여 최종적으로 AP를 분리한다.
6. Elution and regeneration in ion exchange chromatography

이온 교환 크로마토그래피에서 elution은 이온 강도(염 농도)를 높이거나 pH를 변화시켜 단백질을 분리해 내는 방법이다. 강한 강도의 이온이 포함된 완충액으로 컬럼을 세척하는 regeneration 과정을 통해 컬럼의 전체 용량을 다음 실험에 재사용할 수 있다.

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1. Column chromatography

Column chromatography is a powerful analytical technique used to separate and purify a wide range of chemical compounds. It involves the use of a stationary phase, typically a solid adsorbent material packed into a column, and a mobile phase, which is a liquid or gas that flows through the column. The separation of compounds is based on their differential interactions with the stationary phase, which can be influenced by factors such as polarity, size, and charge. Column chromatography is widely used in various fields, including organic chemistry, biochemistry, and environmental analysis, to isolate and purify target compounds from complex mixtures. The technique offers high resolution, versatility, and the ability to scale up for preparative applications. Understanding the principles and practical aspects of column chromatography is essential for researchers and analysts working in diverse areas of chemical and biological sciences.
2. Adsorption chromatography and partition chromatography

Adsorption chromatography and partition chromatography are two distinct modes of column chromatography, each with its own unique principles and applications. Adsorption chromatography relies on the selective adsorption of analytes onto the surface of a solid stationary phase, such as silica gel or alumina. The separation is based on the differences in the strength of interaction between the analytes and the stationary phase, which can be influenced by factors like polarity, polarizability, and hydrogen bonding. Partition chromatography, on the other hand, involves the partitioning of analytes between two immiscible liquid phases, typically an aqueous mobile phase and an organic stationary phase. The separation is driven by the differences in the solubility and distribution of the analytes between the two phases. Both adsorption and partition chromatography have their own advantages and are widely used in various fields, including organic synthesis, natural product isolation, and environmental analysis. Understanding the principles and selecting the appropriate chromatographic mode is crucial for achieving efficient and selective separations.
3. Ion exchange chromatography

Ion exchange chromatography is a powerful separation technique that relies on the reversible exchange of ions between a stationary phase and a mobile phase. The stationary phase is typically a resin or polymer with charged functional groups that can attract and bind to oppositely charged ions in the sample. The separation is based on the differences in the strength of interaction between the analytes and the charged groups on the stationary phase, which can be influenced by factors such as pH, ionic strength, and the charge density of the analytes. Ion exchange chromatography is widely used in various applications, including the purification of proteins, nucleic acids, and other biomolecules, as well as the analysis of inorganic ions and small organic molecules. The technique offers high resolution, selectivity, and the ability to scale up for preparative applications. Understanding the principles and practical aspects of ion exchange chromatography is essential for researchers and analysts working in the fields of biochemistry, analytical chemistry, and environmental science.
4. Alkaline phosphatase (AP)

Alkaline phosphatase (AP) is a ubiquitous enzyme found in various tissues and body fluids, including the liver, bone, intestine, and placenta. It plays a crucial role in numerous physiological processes, such as bone mineralization, nutrient absorption, and signal transduction. AP is commonly used as a biomarker in clinical diagnostics, as its levels can provide valuable information about the health and function of different organs. Elevated AP levels can be indicative of various pathological conditions, including liver disease, bone disorders, and certain types of cancer. Understanding the structure, function, and regulation of AP is essential for interpreting its diagnostic significance and developing effective therapeutic strategies targeting this enzyme. Ongoing research in the field of AP continues to expand our knowledge of its diverse biological roles and its potential as a therapeutic target in various disease states.
5. Purification of alkaline phosphatase

The purification of alkaline phosphatase (AP) is an important process in both research and clinical settings, as it allows for the isolation and characterization of this enzyme for various applications. The purification of AP typically involves a combination of different chromatographic techniques, such as ion exchange, affinity, and size exclusion chromatography. These methods exploit the unique physicochemical properties of AP, such as its charge, substrate-binding ability, and molecular weight, to selectively separate it from other proteins and impurities in the sample. The purified AP can then be used for a variety of purposes, including enzyme activity assays, structural studies, and the development of diagnostic tests or therapeutic interventions. Optimizing the purification process is crucial to obtain high-purity AP with minimal loss of activity, which is essential for accurate biochemical and clinical analyses, as well as for the development of reliable and sensitive AP-based assays and therapeutic applications.
6. Elution and regeneration in ion exchange chromatography

Elution and regeneration are critical steps in the ion exchange chromatography process, as they determine the efficiency and reusability of the ion exchange resin. Elution involves the selective desorption of the target analytes from the resin by using an appropriate eluent, typically a buffer solution with a specific pH, ionic strength, or composition. The choice of eluent depends on the nature of the analytes and the ion exchange mechanism involved. Effective elution ensures the complete recovery of the target compounds while minimizing co-elution of unwanted impurities. Regeneration, on the other hand, involves the restoration of the ion exchange resin's adsorptive capacity by removing any bound ions or contaminants. This is typically achieved by washing the resin with a high-ionic-strength solution or a solution with a different pH. Proper regeneration is crucial for maintaining the resin's performance and extending its lifespan, allowing for repeated use in subsequent purification cycles. Understanding the principles and optimization of elution and regeneration in ion exchange chromatography is essential for developing efficient and cost-effective purification strategies in various fields, including biochemistry, analytical chemistry, and industrial biotechnology.