Cell harvest and protein assay

문서 내 토픽

• 1. Cell harvest

  세포 배양 시 adherent cell과 suspension cell로 구분할 수 있으며, adherent cell을 harvest하는 방법에는 화학적 방법(trypsin, accutase 사용)과 물리적 방법(scraper 사용)이 있다. 이번 실험에서는 물리적 방법인 scraper를 사용하여 adherent cell을 harvest하였다.

• 2. Protein extraction

  세포 내 단백질을 추출하기 위해 lysis buffer와 phosphatase/protease inhibitor를 사용하여 세포막을 부수고 단백질을 추출하였다. 이때 phosphatase/protease inhibitor는 단백질의 분해와 탈인산화를 막아준다.

• 3. Protein assay

  추출한 단백질의 총량을 측정하기 위해 Bradford assay를 사용하였다. 이 방법은 Coomassie Blue 용액과 단백질이 반응하여 흡광도가 변하는 것을 이용한다. 표준 곡선을 만들어 시료의 단백질 농도를 측정하고, 가장 낮은 농도의 시료에 맞추어 다른 시료의 농도를 조정하였다.

• 4. SDS-PAGE 전처리

  단백질의 3차 및 4차 구조를 풀어주기 위해 4X sample buffer를 사용하여 전처리를 진행하였다. 이 buffer에는 TrisHCl, SDS, Glycerol, β-mercaptoethanol, bromophenol blue 등이 포함되어 있어 단백질의 전하를 모두 음전하로 바꾸고 선형으로 만들어준다.

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• 1. Cell harvest

  Cell harvest is a critical step in many biotechnology and pharmaceutical processes, as it involves the separation and collection of cells from a culture or suspension. The choice of cell harvest method can significantly impact the yield, purity, and viability of the final product. Factors such as cell type, culture conditions, and downstream processing requirements must be carefully considered when selecting the appropriate cell harvest technique. Centrifugation, filtration, and affinity-based methods are commonly used, each with their own advantages and limitations. Optimizing the cell harvest process is essential to ensure the efficient and consistent production of high-quality biomolecules and therapeutics.

• 2. Protein extraction

  Protein extraction is a fundamental technique in biochemistry and molecular biology, as it involves the isolation and purification of proteins from complex biological samples. The choice of extraction method can greatly influence the yield, purity, and structural integrity of the target proteins. Factors such as the nature of the sample, the properties of the target protein, and the intended downstream applications must be considered. Common extraction methods include mechanical disruption, chemical lysis, and affinity-based techniques, each with their own strengths and limitations. Optimizing the protein extraction process is crucial to ensure the successful isolation of high-quality proteins for a wide range of research and industrial applications, including enzyme studies, structural analysis, and the development of therapeutic proteins.

• 3. Protein assay

  Protein assays are essential analytical tools used to quantify the concentration of proteins in a sample. Accurate protein quantification is crucial in various fields, such as biochemistry, cell biology, and biotechnology, where the precise measurement of protein levels is required for experimental design, process optimization, and quality control. A wide range of protein assay methods are available, each with their own advantages and limitations in terms of sensitivity, specificity, and compatibility with different sample types and interfering substances. The choice of protein assay depends on factors such as the available equipment, the nature of the sample, and the desired level of accuracy and precision. Careful optimization and validation of the protein assay protocol are necessary to ensure reliable and reproducible results, which are essential for making informed decisions in research, development, and production settings.

• 4. SDS-PAGE 전처리

  SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) is a widely used technique for the separation and analysis of proteins based on their molecular weight. The pre-treatment of samples is a critical step in the SDS-PAGE process, as it ensures the effective denaturation and solubilization of proteins, allowing for their efficient separation and visualization. Factors such as the choice of denaturing agent, the concentration of SDS, the presence of reducing agents, and the incubation time and temperature can all impact the quality and reproducibility of the SDS-PAGE results. Proper sample preparation, including the removal of interfering substances and the optimization of the pre-treatment conditions, is essential to obtain reliable and interpretable protein profiles. Careful attention to the SDS-PAGE pre-treatment steps can lead to improved resolution, better band separation, and more accurate molecular weight determination, which are crucial for various applications, such as protein characterization, identification, and quantification.