Protein separation is a fundamental and indispensable step in the field of protein sequencing. It plays a pivotal
role in facilitating the analysis of complex mixtures of proteins, understanding their functions, and unraveling the
intricacies of biological systems.
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.
This 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-PAGE
SDS-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.
Protein Linearization with SDS
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.
Polyacrylamide Gel Electrophoresis
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.
Protein Separation Mechanism
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-PAGE
To ensure the success of an SDS-PAGE experiment, it's important to follow best practices carefully.
Sample Preparation
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.
Gel Selection
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.
Optimal Running Conditions
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-PAGE
2D-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.
Isoelectric Focusing (IEF)
- 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.
SDS-PAGE
- SDS-PAGE Principle: See the SDS-PAGE section in detail
- Role 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.
Best Practices of 2D-PAGE
IEF Process
Sample Preparation: Prior to IEF, protein samples are typically prepared by solubilizing them in a rehydration buffer
that includes a reducing agent and a detergent. This helps to ensure proper protein solubility and denaturation.
Rehydration: The solubilized protein sample is applied to an immobilized pH gradient (IPG) strip, which serves as the
first-dimensional gel. The strip is then subjected to a rehydration step to allow the proteins to migrate according
to their pI.
IEF Conditions: Careful control of IEF conditions, including voltage and focusing time, is essential for achieving
optimal separation based on charge. The pH gradient and buffer composition should match the pI range of the proteins
being studied.
Sensitive Staining Methods
Staining: After 2D-PAGE separation, proteins are typically visualized using staining techniques such as Coomassie
Brilliant Blue or silver staining. For enhanced sensitivity, especially when dealing with low-abundance proteins,
fluorescent or mass spectrometry-compatible stains may be preferred.
Protein Detection: The choice of detection method, such as fluorescence or chemiluminescence, can significantly
impact the sensitivity of protein visualization. Additionally, using image analysis software for quantification and
comparison of protein spots can enhance the accuracy of results.
Liquid Chromatography (LC)
Principles of LC
Liquid Chromatography (LC) is a widely utilized technique in bioanalysis and separation. It is based on the
principles of distribution and separation of substances in a liquid carrier. Here is an in-depth look at the
principles of liquid chromatography:
Distribution and Equilibrium
At its core, liquid chromatography relies on the distribution of analytes (such as proteins) between two phases: a
stationary phase and a mobile phase. The stationary phase is typically a solid support, like a column packed with
beads, while the mobile phase is a liquid solvent.
Equilibrium: Analytes distribute themselves between the stationary and mobile phases in a dynamic equilibrium. The
extent of distribution depends on factors such as the chemical properties of the analytes, the stationary phase, and
the mobile phase.
Types of Columns
Different types of columns are employed in liquid chromatography, each with its unique separation mechanism:
- Reverse-Phase Chromatography:
Principle: In reverse-phase chromatography, the stationary phase is nonpolar (hydrophobic), while the mobile phase is
polar (usually water with an organic modifier). Polar analytes are retained more by the nonpolar stationary phase
and are eluted later, while nonpolar analytes are eluted earlier.
- Affinity Chromatography:
Principle: Affinity chromatography exploits the specific interactions between a ligand (immobilized on the stationary
phase) and a target analyte. This technique is highly selective, allowing for the isolation and purification of
proteins based on their affinity for a specific ligand.
- Size-Exclusion Chromatography:
Principle: Size-exclusion chromatography separates analytes based on their size and shape. Larger analytes cannot
penetrate the porous stationary phase beads and, therefore, move through the column more rapidly than smaller
analytes.
Diagram of the
liquid chromatography system (Torre et al., 2015).
Protein Separation Mechanism
The separation of proteins in liquid chromatography is primarily influenced by their interactions with the stationary
phase. In reverse-phase chromatography, it's the hydrophobicity of proteins that determines their retention time. In
affinity chromatography, it's the specific binding affinity to the immobilized ligand. In size-exclusion
chromatography, it's the size and shape of the proteins that influence their elution order.
Best Practices of LC
Column Selection
Choose the Right Column: Selecting an appropriate column is crucial. Consider factors such as the nature of the
analytes, their size, and the desired separation mechanism. Different types of columns offer different selectivity.
Mobile Phase and Flow Rate
Optimize Mobile Phase Composition: The choice of mobile phase composition affects analyte retention and separation.
Adjust solvent composition, pH, and buffer strength as needed.
Flow Rate Optimization: Flow rate influences resolution and analysis time. It should be optimized to achieve the
desired separation efficiency without compromising peak resolution.
Detector Sensitivity
Use Sensitive Detectors: Depending on the analytes and their concentrations, choose detectors that offer adequate
sensitivity. Common detectors include UV-Vis, fluorescence, and mass spectrometry detectors.
References
- Mad-Ali, Sulaiman, and Soottawat Benjakul. "Characteristics and properties of goat meat gels and balls as
affected by setting conditions." Food Quality and Safety 3.2 (2019): 129-136.
- Torre, César Aquiles Lázaro de la, et al. "Chromatographic detection of nitrofurans in foods of
animal origin." Arquivos do Instituto Biológico 82 (2015): 1-9.
For research use only, not intended for any clinical use.
