Essential Techniques for Precise Protein Separation in Sequencing
Creators: name: West, Dora
Publisher: name: figshare
Year: 2024
Subjects: classificationCode: 310101
subject: Analytical biochemistry
classificationCode: 310109
subject: Proteomics and intermolecular interactions (excl. medical proteomics)

description: Why Protein Separation is Essential?Complexity of Protein Mixtures: Biological samples, whether they are derived from cells, tissues, or organisms, contain a multitude of different proteins. These proteins vary in terms of size, charge, structure, and function. Without separation, the intricate mixture of proteins would confound any attempt to analyze and study them effectively.Identification and Characterization: To fully understand the role of specific proteins within a biological system, it is imperative to isolate and identify them individually. Protein separation allows researchers to isolate a target protein of interest, enabling subsequent analyses such as mass spectrometry for identification and characterization.Functional Studies: Separation of proteins permits researchers to perform in-depth functional studies. By isolating individual proteins or protein complexes, scientists can investigate their roles in cellular processes, signaling pathways, and disease mechanisms. This information is vital for advancing our knowledge of biology and medicine.Quality Control: In various applications, such as biopharmaceutical production, ensuring the purity of specific proteins is essential. Protein separation techniques enable quality control by isolating and purifying proteins to meet stringent standards.Select ServiceProtein Sequencing ServiceBiopharmaceutical Characterization ServicesProteomics Analysis ServicesThis article delves into three essential protein separation strategies: SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis), and Liquid Chromatography.SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)Principles of SDS-PAGESDS-PAGE is a widely used technique for the separation of proteins based on their molecular weight. This method employs the use of SDS and polyacrylamide gel to linearize proteins and separate them in an electric field. Let's delve into the principles underlying SDS-PAGE.<b>Protein Linearization with SDS</b>Sodium Dodecyl Sulfate (SDS): SDS is an anionic detergent that plays a central role in SDS-PAGE. When proteins are mixed with SDS, the detergent molecules coat the protein molecules. Importantly, SDS binds to proteins in a ratio of approximately one SDS molecule per two amino acids. This binding causes proteins to denature, disrupting their secondary and tertiary structures. As a result, the protein molecules are now uniformly coated with negative charges, proportional to their length. This uniform negative charge effectively eliminates the influence of protein shape or charge on their mobility during electrophoresis.<b>Polyacrylamide Gel Electrophoresis</b>Polyacrylamide Gel: The gel used in SDS-PAGE is made of polyacrylamide, a synthetic polymer that forms a porous matrix when polymerized. The concentration of polyacrylamide in the gel can be adjusted to create different pore sizes, which in turn affects the separation of proteins. Higher acrylamide concentrations yield smaller pores and are suitable for separating smaller proteins.<b>Protein Separation Mechanism</b>Once the protein samples have been linearized with SDS, they are loaded into wells at the top of the polyacrylamide gel. When an electric current is applied across the gel, proteins migrate through the gel matrix towards the positively charged electrode. The migration rate is primarily determined by the molecular weight of the proteins. Smaller proteins move more rapidly through the gel, while larger ones progress more slowly. As a result, the proteins separate according to their molecular weights, with the smallest proteins migrating the farthest from the wells.Best Practices of SDS-PAGETo ensure the success of an SDS-PAGE experiment, it's important to follow best practices carefully.<b>Sample Preparation</b>Sample Denaturation: Prior to loading samples onto the gel, they should be denatured and reduced. This typically involves boiling the samples in the presence of a reducing agent like beta-mercaptoethanol or dithiothreitol (DTT). This step ensures that the proteins remain linearized and denatured.<b>Gel Selection</b>Choosing Gel Concentration: The choice of gel concentration is crucial and depends on the size range of the proteins of interest. For smaller proteins, higher-percentage gels (e.g., 15%) are suitable, while larger proteins may require lower-percentage gels (e.g., 8%). It's essential to choose the appropriate gel to achieve the desired separation.<b>Optimal Running Conditions</b>Buffer System: Ensure the use of an appropriate buffer system. Tris-glycine or Tris-tricine buffers are commonly used for SDS-PAGE. The buffer provides ions necessary for conduction and maintains a stable pH.Voltage and Running Time: The voltage applied during electrophoresis and the duration of the run depend on the size of the gel and the desired separation. Higher voltages can lead to faster runs but may generate excess heat. Carefully adjust the voltage and running time to prevent overheating and distortion of bands.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of proteins in goat meat gels with different setting times in the absence and presence of CaCl2 (Mad-Ali et al., 2019)2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis)Principles of 2D-PAGE2D-PAGE is a powerful technique used for the separation and analysis of complex protein mixtures. It combines two distinct separation mechanisms, isoelectric focusing (IEF) and SDS-PAGE, to achieve high-resolution separation based on both charge and molecular weight.<b>Isoelectric Focusing (IEF)</b>IEF Principle: IEF is the first dimension of 2D-PAGE and separates proteins based on their isoelectric points (pI). Isoelectric point is the pH at which a protein carries no net electrical charge. In IEF, proteins are placed in a pH gradient gel, and an electric field is applied. Proteins migrate within the gel until they reach the pH that matches their pI. At this point, they stop migrating because they no longer experience a net electrical force.Role in Protein Separation: IEF separates proteins according to their charge, placing them along the pH gradient according to their pI. Proteins with different pI values will move to different positions in the gel.<b>SDS-PAGE</b>SDS-PAGE Principle: See the SDS-PAGE section in detailRole in Protein Separation: SDS-PAGE resolves the proteins based on their molecular weight. Proteins that have similar pI values but different molecular weights will be separated from one another.The combination of IEF and SDS-PAGE in 2D-PAGE allows for the separation of highly complex protein mixtures. Proteins are first separated based on their charge in the IEF dimension and then further separated based on their size in the SDS-PAGE dimension. This two-step process results in a high-resolution protein separation, allowing for the visualization of individual proteins even within a complex mixture.