Electrophoresis: Principles, Methods, and Applications PDF

# ELECTROPHORESIS ## What is Electrophoresis? Electrophoresis is a technique that separates molecules based on their charge and size. - **Definition:** The migration of a charged particle through a solution under the influence of an external electrical field. - **Etymology:** The word electropho...

# ELECTROPHORESIS ## What is Electrophoresis? Electrophoresis is a technique that separates molecules based on their charge and size. - **Definition:** The migration of a charged particle through a solution under the influence of an external electrical field. - **Etymology:** The word electrophoresis comes from the Greek words *electro* (electricity) and *phoresis* (movement). **Principle:** - Charged molecules migrate in an electric field. - The rate of migration depends on: - The molecule's net charge - The molecule's size - The molecule's shape - The applied electric current - The formula $v = Eq/F$ describes the velocity of a molecule during electrophoresis: - v = velocity of the molecule - E = electric field (Volt/cm) - q = net charge on molecule - F = frictional coefficient, which depends upon mass and shape of the molecule. - Additionally, these factors affect migration rate: 1. Net charge of the molecule. 2. Size, mass, and shape of the particle. 3. Strength of the electrical field. 4. Properties of the supporting medium. 5. Temperature of operation. ## Factors Affecting Electrophoresis ### Sample Factors - **Charge:** The rate of migration increases as the net charge of the molecule increases. - **Size:** The rate of migration decreases as the size of the molecule increases. This is due to frictional and electrostatic forces. - **Shape:** Molecules with similar charges but different shapes exhibit different migration rates. ### Electric Field Factors - **Voltage:** Increasing the voltage leads to a faster rate of migration. - **Current:** Increasing the current leads to a higher voltage, which also increases the rate of migration. - **Resistance:** Increasing the resistance slows down migration. ### Buffer Factors - **Ionic Strength:** Higher ionic strength increases the rate of migration. - **pH:** The ionization of organic acid increases as the pH increases. ### Supporting Medium Factors - **Adsorption:** Adsorption onto the supporting medium reduces both the rate of migration and resolution of separation. - **Electro-endosmosis:** Charged groups on the surface of the support medium can accelerate the movement of cations and retard the movement of anions. Examples include: - Paper (carboxyl group) - Agarose (sulphate group) ## Electrophoretic Mobility - **Definition:** The rate of migration (cm/sec) per unit field strength (Volts/cm). - **Formula:** μ=Q/6πτη - μ = Electrophoretic mobility - Q = Net charge on the ion - r = Ionic radius of the solute - η = Viscosity of the medium - **Factors:** Electrophoretic mobility is directly proportional to net charge and inversely proportional to molecular size and viscosity of the electrophoresis medium. - **pH Impact:** The pH of the solution affects the mobility of the ion by determining the amount and nature of the charge. - **Suitable Molecules:** Proteins, nucleic acids, nucleotides, and amino acids bear charged polar groups, making them suitable for electrophoresis. - **Carbohydrates:** Carbohydrates carrying no charged groups must be bound to charged groups (like borate or sulfite ions) before electrophoresis can be performed. - **Lipids:** Lipids are not electrophoresed because they are insoluble in the polar solvents required for electrophoretic current. ## Electrophoresis Apparatus - **Components:** 1. Buffer tank: Holds the buffer. 2. Buffer: Provides an optimal pH and ionic strength for the separation. 3. Electrodes: Made of platinum or carbon and conduct current. 4. Power supply: Provides the electrical current. 5. Support media: Provides a matrix for the separation. - **Buffer Selection:** The choice of buffer depends on the nature of the substance being separated. - **Power Supply:** The electricity is supplied at a constant current and voltage. - **Support:** The support on which the separation takes place may contact the buffer directly or by means of wicks. - **Protection:** The entire apparatus is covered to minimize separation. - **Diagram:** A typical electrophoresis apparatus consists of two buffer boxes with baffle plates, electrodes, an electrophoretic support, wicks, a cover and a power supply. ## Support Media - Various types of support media are used in electrophoresis and vary from pure buffer solution in a capillary to insoluble gels (e.g., sheet, slabs, or columns of starch, agarose, or polyacrylamide) or membranes of cellulose acetate. - **Examples:** 1. Filter Paper 2. Cellulose acetate membrane 3. Agar or Agarose gel 4. Starch Gel 5. Polyacrylamide gel ## Buffers - The buffer is a multifunctional component in the electrophoretic process: - **Carries the applied current:** - **Establishes the pH:** - **Determines the electrical charge:** - **Common Buffers for Electrophoresis:** | Intended Separation | Buffer | pH | Ionic Strength (μ) | Composition (g/L) | |---|---|---|---|---| | Proteins | Barbital* | 8.6 | 0.05 | 10.3 Na barbiturate, 1.84 Barbital | | | Barbital | 8.6 | 0.075 | 15.45 Na barbiturate, 2.76 Barbital | | | Rheophor | 8.6 | 0.05 | 10.05 Na barbiturate, 90 ml 0.1 N HCL | | | Phosphate | 7.4 | 0.6 | 0.6 NaH2PO4 • H2O, 2.2 Na2HPO4 | | | Borate | 9.0 | 0.62 | 7.63 Na borate, 0.62 Boric acid | | Mucoproteins | Acetate | 4.5 | 0.1 | 3.51 NaCl, 3.28 g Sodium acetate, pH adjusted with HCl to 4.5 | | | Phosphate | 4.4 | 0.2 | 9.44 g Na2HPO4 | | | Barbituate | 8.6 | 0.1 | 10.3 g citric acid, 20.6 g Na barbiturate | | | Citrate | 4.5 | 0.13 | 3.68 g Barbital, 28.46 g Na citrate | | Amino Acids | Phosphate | 4.6 | 0.15 | 20.4 g KH2PO4 | | | Phosphate | 7.2 | 1/15 M | 2.196 g NaH2PO4 • 2H2O, 8.622 g Na2HPO4 • 2H2O | | | Michaelis | 8.6 | 0.1 | 9.8 g Na barbiturate | | Nucleic Acids | Borate | 9.2 | 0.05 | 3.9 g Na acetate, 60 ml 0.1 N HCL, 3.3 g Na borate | | | Tris Acetate | 7.8 | 24.1 | g Tris base, 8.2 g Na acetate, 1.85 g Na2EDTA • 2H2O, pH adjusted with acetic acid (5 x stock solution) | | | Tris Phosphate | 7.7 | 21.8 g Tris base, 23.4 g NaH2PO4 • 2H2O, 1.85 g Na2EDTA • 2H2O, (5 x stock solution) | - **Ionic Strength Impact:** As the ionic strength of the buffer increases, the proportion of current carried by the buffer increases, and the share of current carried by the sample decreases, slowing down the rate of migration. - **Optimal Ionic Strength:** An ionic strength of 0.05M is preferred for separating proteins or lipoproteins in an electric field. ## Working Procedure of Electrophoresis 1. **Hydration and Placement:** The porous support is hydrated with a suitable buffer and placed between two chambers containing the buffer. 2. **Sample Application:** The sample is applied to the support at the cathode end. The applied current causes components to migrate from the cathode to the anode. 3. **Fixing and Preventing Diffusion:** At the end of the run, the support is removed, and the position of the molecules on the support is fixed with a fixative to prevent sample diffusion. 4. **Visualization:** The separated components are stained to visualize them. 5. **Quantification:** The bands can be quantitated (by elution or by scanning with a densitometer) as the uptake of the dye is proportional to the concentration of the molecule in each band. ## Types of Electrophoresis ### a) Zone Electrophoresis - **Description:** Involves the migration of the charged particle on the supporting media. - **Support Media:** Paper, cellulose acetate membrane, starch gel, or polyacrylamide gel. - **Zone Formation:** Components separated are distributed into discrete zones on the support medium. - **Buffer Saturation:** The supporting media is saturated with buffer solution, and a small volume of the sample is applied as a narrow band. - **Migration Rate:** When potential difference is applied, the components migrate at a rate determined by their electrophoretic mobility. ### b) Moving Boundary/Frontal Electrophoresis - **Principle:** Charged species migrate in a free-moving solution without a supporting medium. - **Advantages:** 1. Biologically active fractions can be recovered without the use of denaturing agents. 2. Minute concentrations of samples can be detected. - **Disadvantages:** 1. Costlier. 2. Requires an elaborate optical system. - **Applications:** 1. Studying the homogeneity of a macromolecular system. 2. Analyzing complex biological mixtures. ### Zone Electrophoresis Types #### 1) Paper Electrophoresis - **Description:** A form of electrophoresis carried out on filter paper. - **Applications:** Separating small charged molecules, such as amino acids and small proteins. - **Diagram:** A simplified paper electrophoresis apparatus. - **Components:** - **Filter Paper:** Acts as the stabilizing medium. Whatman, cellulose acetate, or chromatographic paper can be used. - **Apparatus:** The apparatus consists of a power back, an electrophoretic cell containing electrodes, buffer reservoirs, a support for the paper, and a transparent insulating cover. - **Sample Application:** The sample can be applied as a spot (about 0.5 cm in diameter) or as a uniform streak. - **Electrophoretic Run:** The current is switched on after the sample is applied to the paper, and the paper is equilibrated with the buffer. The type of buffer used depends on the type of separation being performed. Once the run is complete, the paper is dried in vacuum oven. - **Detection and Quantitative Assay:** To identify unknown components in the resolved mixture, the electrophoretogram can be compared to another electrophoretogram, which includes standard components. Physical properties like fluorescence, UV absorption, or radioactivity are exploited for detection. #### 2) Gel Electrophoresis - **Description:** A technique used for separating DNA, RNA, or protein molecules according to their size and electrical charge. - **Gel Composition:** The gel is a cross-linked polymer. Its composition and porosity are chosen based on the specific weight and porosity of the target molecules. - **Diagram:** A setup for gel electrophoresis. - **Gel Types:** 1. Agarose gel 2. Polyacrylamide gel 3. Sephadex gel #### a) Agarose Gel Electrophoresis - **Description:** A highly purified uncharged polysaccharide derived from agar. - **Applications:** Separating macromolecules such as nucleic acid, larger proteins, and protein complexes. - **Preparation:** Agarose is dissolved in boiling water and allowed to cool to 40°C. - **Characteristics:** Agarose gels are fragile due to the formation of weak hydrogen bonds and hydrophobic bonds. - **Diagram:** A simple agarose gel electrophoresis setup. #### b) SDS-PAGE Electrophoresis - **Description:** Sodium dodecyl sulfate polyacrylamide gel electrophoresis. - **SDS Function:** SDS is an anionic detergent that binds strongly to and denatures proteins. This denaturation ensures that protein separation is based solely on size, not shape. - **Charge Impact:** Protein-SDS complexes carry a negative charge, so they move towards the anode. - **Separation and Molecular Mass:** The separation is based on the size of the particle, and it can be used to determine the relative molecular mass of the protein. - **Applications:** 1. Protein purification. 2. Determining the molecular weight. 3. Quantifying proteins. 4. Blotting applications. - **Process of SDS-PAGE:** 1. **Boiling:** Samples are boiled for 10 minutes to denature proteins. Typically, a reducing agent like B-mercaptoethanol is included in the protein sample buffer to break disulfide bonds. 2. **Gel Assembly:** The gel is assembled into the electrophoresis apparatus. 3. **Buffer Addition:** The buffer solution is poured into the chamber. 4. **Sample Loading:** Samples are loaded into the wells of the gel. 5. **Electrophoresis:** Electrophoresis is run by connecting the apparatus to a power supply. 6. **Visualization:** Bands are visualized under a UV light or with a staining technique. - **Diagram:** A visual representation of SDS-PAGE, showcasing sample migration through the gel. #### 3) Thin Layer Electrophoresis - **Description:** Electrophoretic studies can be carried out in thin layers of silica, kieselguhr, or alumina. - **Advantages:** 1. Less time-consuming 2. Good resolution - **Application:** Used in two-dimensional electrophoretic-chromatographic studies of proteins and nucleic acids hydrolysates. #### 4) Cellulose Acetate Electrophoresis - **Description:** Cellulose acetate contains 2-3 acetyl groups per glucose unit. Its adsorption capacity is less than that of paper. Cellulose acetate provides sharper bands and a good background for staining glycoproteins.. - **Advantages:** 1. No tailing of proteins 2. Gives sharp bands and good resolution - **Disadvantages:** 1. Expensive 2. The presence of carboxylic residue can cause induced electro-osmosis. - **Diagram:** A visualization of cellulose acetate electrophoresis. ## Moving Boundary Electrophoresis - **Instrumentation:** A U shaped glass cell with electrodes placed on the limbs. The sample solution is introduced at the bottom or through a side arm. The apparatus is placed in a constant temperature bath at 40°C. Detection is done by measuring refractive index throughout the solution (Schlieren optical system). - **Diagram:** A depiction of the U shaped cell used for moving boundary electrophoresis. ### 1) Capillary Electrophoresis: - **Description:** A capillary tube placed between two buffer reservoirs. An electric field is applied, and separation relies on electrophoretic mobility and electro-osmosis. - **Sample Introduction:** A defined volume of analyte is introduced into the capillary. One buffer reservoir is replaced with a sample vial. - **Detection:** Electrophoretic separation is measured using a detector. - **Band Broadening:** The joule heating effect is removed using narrow bore tubes, which decreases band broadening. This leads to faster separations than gel electrophoresis. - **Tube Dimensions:** Capillary electrophoresis uses tubes with diameters of 20-100µm and lengths of 20-100cm. - **Gel Use:** CE can be performed with or without a gel. - **Diffusion:** Longitudinal diffusion is the main source of band-broadening in capillary electrophoresis. - **Efficiency:** Higher electric fields lead to high efficiency and narrow peaks because the analyte migrates faster. - **Migration Time:** All analytes travel the same distance, but the migration time is measured for that distance to determine the identity of the analyte. - **Peak Area/Height Correlation:** The amount of the analyte is related to the peak area or height. - **Diagram:** A capillary electrophoresis apparatus depicting the flow of the sample through the tube to the detector where the data is measured. ### 2) Isotachophoresis - **Description:** Isotachophoresis is a technique that relies on *Iso* (equal), *tachos* (speed) and *phoresis* (migration). It utilizes a potential gradient to separate molecules. - **Applications:** Separating smaller ionic substances. - **Migration:** Molecules migrate adjacent to each other with contact, but they do not overlap. - **Sample Preparation:** The sample *is not* mixed with the buffer prior to running isotachophoresis. - **Current Flow:** The current flow is entirely carried by the sample ions. - **Application:** Separating small anions and cations. ### 3) Isoelectric Focusing - **Description:** Also known as electro-focusing, this technique separates molecules based on differences in their isoelectric points. - **Applications:** 1. Human genetic lab 2. Separating iso-enzymes 3. Research in enzymology and immunology - **Amphoteric Substances:** Isoelectric focusing is suitable for separating amphoteric substances. - **Protein Separation:** Often performed on proteins in a gel. - **Charge Importance:** The overall charge on the molecule is a function of the pH of its surroundings. - **High Resolution:** Isoelectric focusing offers high resolution. ### 4) Immuno-electrophoresis - **Description:** This technique studies antigen-antibody reactions using an electric field. - **Antibody Separation:** Antibodies are electrophoretically separated, and antigens diffuse towards them, forming precipitin arcs. - **Applications:** - Detecting the presence of antibodies. - Determining the blood levels of three major immunoglobulins: - IgM - IgG - IgA - **Process:** - **Antigen Separation:** Ag molecules are separated based on their electrical charge by electrophoresis. - **Antibody Placement:** Antibodies are placed in a trough cut in the agar. - **Precipitin Arc Formation:** Antibodies diffuse towards antigens, creating precipitin arcs. - **Interpretation:** Precipitin arcs represent individual antigens. - **Diagram:** A visual representation of the steps involved in immuno-electrophoresis. ## Applications of Electrophoresis 1. DNA Sequencing 2. Medical Research 3. Protein Research/Purification 4. Agricultural testing 5. Separating organic acids, alkaloids, carbohydrates, amino acids, alcohols, phenols, nucleic acids, and insulin. 6. Food industry 7. Biochemical and clinical fields, particularly in studies of protein mixtures, like blood serum and haemoglobin, and antigen-antibody interactions. 8. Studying iron binding to serum proteins in combination with auto-radiography. 9. Analyzing terpenoids, steroids, and antibiotics. 10. Testing the purity of thyroid hormones using zone electrophoresis. 11. Separating free insulin from plasma proteins using paper chromato-electrophoresis. 12. Diagnosing various diseases of the kidney, liver, or cardiovascular system. 13. Separating scopolamine and ephedrine using a buffer with a pH of 4.2. 14. Separating carbohydrates and vitamins. 15. Quantitative separation of cellular entities, such as antibiotics, red blood cells, enzymes, etc.