Introduction
In 1937, Swedish biochemist Arne Tiselius demonstrated that charged particles can be separated based on their charge using an electrical field. Biomolecules such as proteins, peptides, nucleic acids, and nucleotides also possess electrical charges and migrate towards either the anode or cathode based on their net charge in an electric field. This process is known as electrophoresis, which involves the migration of electrically charged molecules in response to an electric field.[1]
Tiselius used a liquid medium that had less resolution due to the effect of gravity and diffusion. Electrophoresis uses solid support media with buffers to overcome these obstacles. Molecules with similar charge, mass, shape, and size tend to move together and are separated into distinct bands or zones. Common solid support media include Whatman filter paper, agarose, cellulose acetate, and polyacrylamide.[1]
General Components of an Electrophoresis Apparatus
An electrophoresis apparatus consists of several key components, each with a specific function that separates charged molecules (see Image. Schematic Diagram of an Electrophoresis Apparatus).
Buffer: Carries the electric current and maintains the pH of the medium.
Wicks: Connect the support medium with the buffer to complete the circuit.
Support medium: Serves as the matrix in which the separation of molecules takes place.
Cover: Reduces evaporation of buffer and prevents contamination during the electrophoretic run.
Power supply: Provides an electrical field for the movement of charged particles.
Densitometer: Quantification of separated bands is performed by comparing the optical density of the bands.
Factors Affecting the Electrophoretic Mobility of a Molecule
Size, shape, and net charge of the molecule: Mobility is inversely proportional to the size of the molecule and directly proportional to the net charge of the molecule. Globular proteins have compact structures and faster mobility compared to fibrous proteins of similar molecular weight.[2]
Particles with a negative charge (anions) always move in the direction of the positive pole, whereas particles with a positive charge (cations) always move in the direction of the negative pole. When performing gel electrophoresis, the positive pole refers to the anode, whereas the negative pole refers to the cathode. As a result, charged particles move to the nodes that are appropriate for them. In gel electrophoresis, anions migrate from the cathode (−) to the anode (+).[2]
Strength of the electrical field: Mobility is proportional to the potential gradient (voltage) and inversely proportional to resistance.
Buffer: Buffer functions to carry the current and maintain the pH of the medium. The optimum ionic strength of the buffer is necessary as higher ionic strength increases the share of current carried by buffer ions, slowing down the sample migration and generating heat that leads to increased diffusion of separation bands. The low ionic strength of the buffer also reduces resolution due to reduced overall current passing through the medium.
The ionization of molecules, such as proteins and amino acids, depends on the pH of the medium. Alteration in the pH of the medium can alter the direction and velocity of migration.[2]
Supporting medium: A medium with affinity for sample molecules can impede their migration rate and reduce the resolution of separation. The pore size of the support medium is inversely proportional to the gel concentration; therefore, adjusting pore size according to the properties of the molecule of interest is necessary for optimal resolution.
Fixed groups, such as sulfate, get ionized and acquire a negative charge at alkaline or neutral pH. When an electric field is applied, HO ions associated with these negatively charged groups start migrating toward the cathode. This movement hinders sample movement towards the anode and can reduce separation resolution. This phenomenon is known as electroendosmosis. To minimize its effects, ultrapure agarose gel with low sulfate content can be used.[3]
Types of Support Medium
Different support media and buffers are used to effectively separate various molecules.
Whatman filter paper: Whatman filter paper serves as a support medium. As it requires a long runtime (12-16 h) and low voltage for separation, the resolution is poor due to the increased diffusion of the separated analytes.[4][5]
Cellulose acetate: Cellulose acetate membranes are a preferred solid medium, as they require less runtime (<1 h). As a result, the resolution of separated bands is significantly superior to that of paper electrophoresis. Although expensive, they are widely used for separating lipoproteins, proteins, enzyme isoforms, and hemoglobin variants due to superior resolution and less interaction with analytes in a sample.[5][6]
Agarose gel: Agarose is a type of heteropolysaccharide that forms a viscous solution when dissolved in a hot buffered solution (50-55 °C) but solidifies into a gel upon cooling. This support medium separates serum proteins, hemoglobin, nucleic acids, and polymerase chain reaction (PCR) products. Fixed sulfate groups present in agarose can reduce the resolution of bands due to increased electroendosmosis, which can be prevented using ultrapure agarose gel with low sulfate content.[5][7]
Polyacrylamide gel: Polyacrylamide gel is formed by polymerizing acrylamide and bis-acrylamide in the presence of ammonium persulfate, N, N, N’, N’-tetramethylethylenediamine, and riboflavin under ultraviolet rays. The pore size of the gel can be precisely controlled by adjusting the concentration of monomers. This gel can be used for various analytes, such as proteins, peptides, nucleic acid, and nucleotides, providing excellent resolution due to better molecular sieving and minimal interaction of sample molecules with the matrix.[5][8]
When a protein solution is briefly boiled in sodium dodecyl sulfate (SDS) and mercaptoethanol, the proteins in the solution become denatured and acquire a uniform negative charge, which masks their native charge. This process produces polypeptide chains with a constant charge-to-mass ratio with a uniform shape. In this condition, electrophoretic mobility depends on the number of amino acids and the mass of the polypeptide chains.[5][9]
Other Variants of Electrophoresis
Isoelectric focusing: The gel matrix is filled with ampholytes (positive and negative charge molecules), forming a pH gradient. When the electricity is applied, molecules migrate towards their isoelectric pH. The mobility of sample molecules stops at their respective isoelectric pH, where the net charge on the sample molecule is zero. Isoelectric focusing can provide excellent resolution and fractionation of serum proteins and hemoglobin variants.[5][10]
Immunoelectrophoresis and immunofixation electrophoresis: Initially, proteins are separated on the agarose gel. Wells are created after separation, and specific antibodies against the target molecules are added to them. Bands of precipitation are formed from an antigen-antibody reaction, which signifies the presence of a specific protein in the sample. This method is used to identify the abnormal elevation of gamma-globulin fractions and free light chains in patients with suspected monoclonal or polyclonal gammopathy.[11]
High-voltage electrophoresis: This technique uses a higher voltage range of 400 to 2000 V for separation compared to the standard 250 V, resulting in high-speed separation with good resolution and relatively less diffusion. High-voltage electrophoresis is commonly used to separate proteins, hemoglobin, and nucleotides.[5]
Pulsed-field electrophoresis: Separation of long nucleotide fragments with good resolution is challenging with conventional electrophoresis. In pulsed-field electrophoresis, the current is passed in 2 different directions alternately, which leads to the movement of fragments in 2 directions, resulting in good separation with optimal resolution.[12]
Capillary electrophoresis: A capillary tube with a minimal diameter, filled with a buffer solution, ampholytes, or gel, serves as the support medium. Due to the availability of a higher surface area for heat dissipation, very high voltage can be applied for speedy separation and better resolution. Separated fractions can be quantified simultaneously as they pass through the detector during the electrophoretic run.[13]
Two-dimensional electrophoresis: Isoelectric focusing is performed to separate the analytes based on their isoelectric pH. The gel containing the separated analytes is then subjected to SDS-polyacrylamide gel electrophoresis at a 90° angle to the isoelectric focusing run. Molecules with similar molecular weights can be separated using this method due to differences in their isoelectric pH.[5][10]
Specimen Requirements and Procedure
Specimen requirements and processing vary depending on the type of electrophoresis and disease involved. Serum, plasma, whole blood, and hemolysate are the most commonly used biological specimens in diagnostic laboratory setups. Nucleic acids or protein extracts from tissue or cellular lysates, as well as products of PCR and sequencing experiments, are the specimens used in research laboratories focused on molecular biology, genomics, and proteomics.[5]
For accurate protein electrophoresis results, proper specimen handling is critical. Repeated freezing and thawing of specimens should be avoided, as this can cause protein degradation, denaturation, or aggregation, which may alter electrophoretic mobility and interfere with interpretation. In specimens with low protein concentration, such as urine and cerebrospinal fluid, protein analysis by electrophoresis requires prior concentration of the sample. Techniques such as ultracentrifugation, dialysis, ultrafiltration, or lyophilization can be used to increase protein yield, thereby enhancing the resolution and sensitivity of protein bands on electrophoresis.
Testing Procedures
Sample details are confirmed using the test requisition form, followed by initial processing in accordance with procedural requirements.
Serum or plasma can be separated from plain or anticoagulant-containing vials using centrifugation. Serum and plasma are used to assess and quantify protein fractions to diagnose disorders related to their synthesis or disposal. Hemolysate can be prepared using buffers and whole blood specimens from the anticoagulant-containing vial. Hemolysate is used to assess and quantify hemoglobin fractions, aiding in the diagnosis of hemoglobinopathies and thalassemia.[14]
Nucleic acid extracts and PCR products can be used after mixing them with sucrose, buffer, and tracking dye. After setting up the instrumentation and gel, the sample is applied, and the electrical supply is started. When tracking dye completes approximately 80% of the run on the gel, the electrical supply is turned off, and the separation of bands is quantified using densitometry.[14][15]
Interfering Factors
The major interfering factors in electrophoresis include heat, nonspecific adsorptive groups on the support medium, and electroendosmosis. As the current and duration of electrophoresis increase, the gel's temperature increases due to heat dissipation. The heat increases the random motion of the molecules in the medium, reducing the sharpness or resolution of the separated bands.
Nonspecific adsorptive groups on the support medium can bind analytes, hindering their mobility across the gel. As described earlier, electroendosmosis generates ion flow opposite to the direction of the analyte motion, leading to a reduction in resolution.[16]
Results, Reporting, and Critical Findings
After the electrophoresis run, the gel is stained for the analyte of interest. After incubation with the staining solution, excess stain is removed by treating the gel with the de-staining solution.
After staining, the gel is visualized using a suitable wavelength of light, and the optical density of each separated band is measured using densitometry. The optical density of each separated band is directly proportional to the concentration of stained analyte present in that band. The report contains the percentage proportion of each stained analyte in the sample.
Table 1. Commonly Used Stains for the Detection of Various Analytes in Electrophoresis.
References for the table.[17][18]
An abnormal electrophoretic pattern observed in serum or hemoglobin electrophoresis can prompt clinicians to focus on identifying an abnormal fraction of protein or hemoglobin in the patient's sample. Careful analysis of these patterns can aid in diagnosing underlying disorders that result in the presence of abnormal analytes or the absence of typical analytes in the case scenario.
Clinical Significance
The electrophoresis apparatus shows abnormal hemoglobin electrophoretic patterns in various hemoglobinopathies and thalassemia (see Image. Hemoglobin Electrophoresis Patterns). Comparing abnormal fractions with the control sample helps clinicians narrow down the diagnosis of hemoglobinopathies and thalassemia.
Table 2. Interpretation of Hemoglobin Electrophoretic Patterns.
References for the table.[19][20]
The presence of abnormal bands or attenuation of the normal band in serum protein electrophoresis can provide clinicians with valuable insight into the ongoing disease process.
Table 3. Main Components of Serum Protein Electrophoresis Zones at pH 8.6.
Reference for the table.[21]
Table 4. Conditions Associated with Abnormalities in Serum Protein Electrophoretic Patterns.
References for the table.[21][22]
The electrophoresis technique can also identify abnormally elevated or decreased enzyme isoforms. Specific enzyme patterns are associated with various clinical conditions, depending on the tissue or organ involved, which aids clinicians in diagnosis and treatment planning.
Table 5. Tissue Origins of Different Isoforms of Clinically Significant Plasma Non-Functional Enzymes.
The utility of electrophoresis is not limited to diagnostics. Electrophoresis is widely used for research in genomics and proteomics. Techniques such as restriction fragment length polymorphism, nucleotide sequencing, next-generation sequencing, southern blotting, and western blotting all incorporate electrophoresis as one of their steps. DNA fingerprinting, a technique employed by forensic experts, compares DNA obtained from crime scenes with that of suspects or victims. Additionally, DNA fingerprinting is used to confirm the biological parents of a child in cases of dispute.[26]
Quality Control and Lab Safety
Commercially available control samples are widely used in hemoglobin, serum protein, and nucleic acid electrophoresis to compare the migration of analytes of interest.
Caution must be exercised throughout the electrophoresis procedure, including gel preparation, buffer preparation, apparatus setup, electrophoresis run, staining, and analyte visualization. Monomers used in the preparation of polyacrylamide gels are carcinogenic. If contacted, the catalyst used in polyacrylamide gel preparation can cause skin damage related to free radicals. None of the solutions should be mouth-piped. Barbital buffer containing sodium barbiturate is a known central nervous system depressant. Ethidium bromide, used in nucleic acid staining, is a known carcinogen. Direct exposure of the eye to ultraviolet rays during the visualization of the gel can cause severe damage to the eye.[27]
Enhancing Healthcare Team Outcomes
Diagnosing medical conditions through electrophoresis is most effective when carried out by an interprofessional team that includes specialists such as internal medicine physicians, biochemists, laboratory medicine experts, and laboratory technicians. Each team member contributes unique expertise, ensuring a comprehensive approach to patient assessment. Internal medicine specialists consider the patient's clinical history and presentation, whereas biochemists provide insights into the biochemical mechanisms underlying various conditions. Laboratory technicians play a vital role in accurately preparing samples and conducting electrophoresis assays, which are essential for obtaining reliable results.
Furthermore, correlating clinical findings with electrophoresis patterns allows clinicians to narrow down the differential diagnosis. By integrating the patient's medical history, physical examination results, and additional laboratory investigations, clinicians can identify specific disorders that may present with similar electrophoretic abnormalities. A precise diagnosis enables healthcare providers to develop targeted treatment plans that address the underlying pathology, ultimately enhancing patient outcomes. This collaborative and systematic approach highlights the importance of teamwork in improving diagnostic accuracy and delivering personalized care to patients.
