Electrophoresis

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. 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. An electrophoresis apparatus consists of several key components, each with a specific function that separates charged molecules (see  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. 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. 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 (+). 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. 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. 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. 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. 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. Polyacrylamide gel: Polyacrylamide gel is formed by polymerizing acrylamide and bis-acrylamide in the presence of ammonium persulfate, , , ’, ’-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. 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. 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. 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.  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. 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.  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. 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.