Tools
Electrophoresis
Introduction
In 1937, Swedish biochemist Arne Tiselius demonstrated that charged particles could 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 anode or cathode based on their net charge in an electric field. Electrophoresis is the migration of electrically charged molecules under the effect of the electrical 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. Whatman filter paper, agarose, cellulose acetate, and polyacrylamide are solid support mediums.[1]
General Components of an Electrophoresis Apparatus
The 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 current and maintains the pH of the medium.
- Wicks: Connects support medium with buffer to complete the circuit.
- Support medium: Provide the matrix in which separation 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 comparison of the optical density of bands.
Factors Affecting 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 cathode (−) to 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 having affinity for the molecules in samples can hinder the rate of migration and can decrease the resolution of separation. The pore size in the support medium is inversely proportional to the gel concentration. Adjusting pore size according to the properties of a molecule of interest is necessary for optimum resolution.
- Fixed groups such as sulfate get ionized and acquire a negative charge at alkaline or neutral pH. Applying the electrical field, 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 mediums and buffers are used to effectively separate various molecules.
Whatman filter paper: Whatman filter paper is a support medium. As it requires long run-time (12-16 hours) and low voltage for separation, the resolution is poor due to the increased diffusion of separated analytes.[4][5]
Cellulose acetate: Cellulose acetate membrane is a preferred solid media as it requires less run-time (<1 hour). Due to this, the resolution of separated bands is far superior to paper electrophoresis. Although expensive, it is 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 as a gel on cooling down. 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]
If a protein solution is boiled briefly in sodium dodecyl sulfate (SDS) and mercaptoethanol, the proteins in the solution get denatured and acquire a uniform negative charge that masks their native charge of the protein. 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 molecules of interest are added. Bands of precipitation are formed from 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 relatively higher voltage (400 to 2000 Volts) instead of 250 Volts for separation, 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 alternatively, which leads to the movement of fragments in 2 directions, giving good separation with optimum resolution.[12]
Capillary electrophoresis: A minimal-diameter capillary tube filled with buffer solution, ampholytes, or gel is used as a 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 separated analytes is then subjected to SDS-polyacrylamide gel electrophoresis in the direction of 90° to the isoelectric focusing run. Molecules having similar molecular weight can be separated through this method due to differences in their isoelectric pH.[5][10]
Specimen Requirements and Procedure
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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 acid or protein extracts from tissue or cellular lysates and products of PCR and sequencing experiments are the specimens used in research laboratories focused on molecular biology, genomics, and proteomics.[5]
Testing Procedures
Confirmation of sample details with the test requisition form is performed, followed by initial processing according to procedural requirements.
Serum or plasma can be separated using centrifugation from plain or anticoagulant-containing vials. 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 to diagnose 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 a run of approximately 80% of 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 are 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 with the suitable wavelength of light, and the optical density of each separated band is quantified by densitometry. The optical density of each separated band is proportional to the concentration of stained analyte present in that band. The report contains the percentage proportion of each stained analyte in the sample. The following table shows stains used for various analytes.[17][18]
| Analyte | Stain |
| Amino acids | Ninhydrin |
| Proteins |
Ponceau S, Coomassie Blue G250, Silver stain, Amido black |
| Lipoproteins | Sudan Black B, Oil Red O |
| Glycoproteins | Periodic Acid-Schiff stain |
| Hemoglobin | Ponceau S, O Dianisidine, Ferricyanide |
| Nucleic acid | Silver stain, Ethidium bromide |
An abnormal electrophoretic pattern of serum or hemoglobin electrophoresis can alert the clinician to emphasize the identification of an abnormal fraction of protein or hemoglobin in the patient's sample. Critical analysis of electrophoretic patterns can help the clinician diagnose an underlying disorder that led to an abnormal analyte or the absence of a typical analyte in the case scenario.
Clinical Significance
The electrophoresis apparatus shows abnormal hemoglobin electrophoretic patterns in various hemoglobinopathies and thalassemia (see Image. Hemoglobin Electrophoresis Patterns). Comparison of abnormal fractions with the control sample helps the clinician narrow the diagnosis of hemoglobinopathies and thalassemia. The following table shows the interpretation of hemoglobin electrophoretic patterns.[19][20]
| Lane No. | Observation and Comment |
| 1 | Electrophoretic pattern of artificial control sample—This pattern is used to compare the position of hemoglobin fractions in the patient's sample. |
| 2 | The electrophoretic pattern of a normal adult shows a major fraction of HbA with traces of HbF and HbA2. |
| 3 | The electrophoretic pattern of normal cord blood shows a major fraction of HbF with a trace of HbA. HbA2 may or may not be found in the normal cord blood samples. |
| 4 | In the electrophoretic pattern of a patient with sickle cell disease, both the genes of the beta-globin chain carry a mutation. The replacement of glutamic acid with valine at the sixth position of the beta-globin chain leads to a reduction in the net charge on the molecule, leading to abnormal hemoglobin, HbS, with less mobility compared to HbA. The major hemoglobin fraction in such cases is HbS with slightly elevated or normal HbF and HbA2. |
| 5 | The electrophoretic pattern of a patient with sickle cell trait shows that 1 of 2 beta-globin chain genes has a mutation that forms HbS. In this case, the presence of HbS and HbA, HbF, and HbA2 are detected. |
| 6 | The electrophoretic pattern of a patient with HbSC disease shows that a mutation in the beta-globin chain results in the formation of HbS in 1 gene product and HbC in another. The replacement of glutamic acid by lysine at the sixth position of the beta-globin chain leads to the formation of HbC. In such cases, the presence of HbS and HbC is detected along with the presence of HbF and HbA2. HbA is remarkably absent in such patients with HbSC disease. |
| 7 | The electrophoretic pattern of a patient with beta-thalassemia minor shows that a mutation leads to reduced synthesis of the beta-globin chain in one of the beta-globin genes. Elevated HbF and HbA2 fractions, along with the presence of diminished or normal HbA fractions, signify the diagnosis of beta-thalassemia minor. |
| 8 | The electrophoretic pattern of a patient with beta-thalassemia major shows that a mutation leads to reduced beta-globin chain synthesis in both the beta-globin genes. In such cases, marked elevated HbF and HbA2 are observed with the absence or minimal HbA. |
| 9 | Patients with silent alpha thalassemia and alpha thalassemia minor may not be detected through electrophoresis as they have a regular electrophoretic pattern. Malfunctioning 1 or 2 out of 4 alpha-globin genes does not impede the synthesis of HbA, as other normal alpha-globin genes can take over the function. |
| 10 | The electrophoretic pattern of a patient with HbH disease shows that 3 of 4 alpha-globin genes . In this case, the decline in HbA levels is found along with the presence of HbH (tetramer of beta chain) or Hb Bart's (tetramer of gamma chain) with relatively faster mobility than HbA. The decline in HbF and HbA2 levels is also detected in such cases of HbH disease. |
| 11 | The electrophoretic pattern of a patient with alpha thalassemia major shows that all 4 alpha globin genes malfunction. Such cases of alpha thalassemia major show the absence of HbA, HbF, and HbA2, along with the presence of HbH and Hb Bart's in hemoglobin electrophoresis. |
The presence of abnormal bands or attenuation of the normal band in serum protein electrophoresis can provide insight to the clinician regarding the ongoing disease process. The following table shows the main content of the zone of serum protein electrophoresis (pH 8.6).[21]
| Zone | Content |
| Albumin | Albumin |
| Albumin-Alpha-1-Interzone | High-density lipoprotein, Alpha-fetoprotein |
| Alpha-1 | Alpha-1-antitrypsin, AFP, alpha-1-glycoprotein, thyroid-binding globulin, and transcortin |
| Alpha-1-Alpha-2-Interzone | Alpha-1-antichymotrypsin, vitamin D–binding protein |
| Alpha-2 | Ceruloplasmin, alpha-2-macroglobulin, haptoglobin |
| Alpha-2-beta-interzone | Pre-beta-lipoprotein |
| Beta | Transferrin, complement protein 3 (C3), beta-lipoprotein—sometimes IgA and IgM |
| Gamma | Mainly IgG. Also, IgA, IgM, IgD, and IgE |
The following table outlines conditions associated with abnormalities in serum protein electrophoretic patterns.[21][22]
| Proteins | Conditions Causing Increase | Condition Causing Decrease |
| Albumin | Hemoconcentration due to dehydration | Burns, nephrotic syndrome, protein-losing enteropathy, impairment of liver functions, chronic infections, malnutrition, and hemodilution in pregnancy |
| Alpha-1-globulins | Pregnancy | Alpha-1-antitrypsin deficiency |
| Alpha-2-globulins | Nephrotic syndrome, corticosteroid therapy | Malnutrition, liver failure, Wilson’s disease |
| Beta globulins | Iron deficiency anemia, obstructive jaundice, third trimester of pregnancy, hypercholesterolemia type 2a | Malnutrition |
| Gamma globulins |
Chronic infections, amyloidosis, lymphoma, leukemia, collagen vascular diseases Sharp bands in the gamma-globin region can be observed in monoclonal gammopathies such as multiple myeloma, solitary plasmacytoma, heavy chain disease, and plasma cell leukemia. |
Agammaglobulinemia, hypogammaglobulinemia |
The electrophoresis technique can also identify abnormally elevated or decreased enzyme isoforms. Specific enzyme patterns are associated with various clinical conditions based on tissue or organ involvement, aiding clinicians in diagnosis and treatment planning. The following table outlines the tissue origins of different isoforms of clinically significant plasma non-functional enzymes.
| Enzyme | Isoforms | Tissue of Origin |
| Lactate dehydrogenase (LDH) [23] | LDH 1 | Cardiac muscle |
| LDH 2 | RBCs, Brain | |
| LDH 3 | Brain, leukocytes | |
| LDH 4 | Liver, leukocytes | |
| LDH 5 | Skeletal muscles | |
| Alkaline phosphatase (ALP) [24] | Alpha-1 ALP | Epithelial cells of biliary canaliculi |
| Alpha-2 ALP (heat labile) | Liver | |
| Alpha 2 ALP (heat stable) | Placenta | |
| Pre-beta ALP | Bone | |
| Creatine kinase (CK) [25] | CK-BB | Brain |
| CK-MB | Cardiac muscle | |
| CK-MM | Skeletal muscle |
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. In addition, DNA fingerprinting is used to confirm the biological parents of a child in case of a dispute.[26] RBCs, red blood cells.
Quality Control and Lab Safety
Commercially available controls are used to compare the movement of the analyte of interest and are widely used for hemoglobin, serum protein, and nucleic acid electrophoresis.
Caution is necessary when preparing electrophoresis gel, buffer preparation, setting up apparatus, running electrophoresis, staining, and visualization of the analyte. Monomers used in polyacrylamide gel preparation are carcinogenic. If contacted, the catalyst used in polyacrylamide gel preparation can cause free radical-related damage to the skin. None of the solutions should be mouth-pipetted. 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 are vital in accurately preparing samples and conducting electrophoresis assays, which is crucial 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 improving patient outcomes. This collaborative and systematic approach highlights the importance of teamwork in enhancing diagnostic accuracy and delivering personalized care to patients.
Media
(Click Image to Enlarge)
Hemoglobin Electrophoresis Patterns
Contributed by Amit Sonagra, MD
(Click Image to Enlarge)
Schematic Diagram of an Electrophoresis Apparatus
Contributed by Amit Sonagra, MD
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