Electrophoresis Lecture Notes

Electrophoresis

Electrophoresis is a biochemical analytic technique used to analyze macromolecules such as proteins, nucleic acids, and lipoproteins. It is also used to separate mixtures of macromolecules and determine their molecular weights.

Basic Principles

• Movement of Charged Molecules: Electrophoresis involves the movement of charged molecules (ions) under the influence of an applied electric field.

• Separation Basis: Macromolecules are separated based on their charges in a medium. Size and shape also play a role to a lesser extent.

• Purity Determination: The technique can determine the degree of purity of a macromolecule.

Factors Affecting Migration Rate

The rate of electrophoretic migration of charged molecules depends on several factors:

1. Electric Field Strength, Size, and Shape:

   • The strength of the electric field influences the force exerted on the molecules.

   • The size and shape of the molecules affect their mobility through the medium.

2. Relative Hydrophobicity of the Sample:

   • Hydrophobicity influences the interaction of the molecules with the buffer and the supporting medium.

3. Ionic Strength and Temperature of the Buffer:

   • Ionic strength affects the electric field and the charge of the molecules.

   • Temperature influences the viscosity of the buffer and the mobility of the molecules.

4. Molecular Weight of the Macromolecule:

   • Larger molecules have more difficulty moving through the supporting medium (e.g., gel).

   • Smaller molecules have faster mobility.

5. Net Charge Density on the Macromolecule:

   • The net charge and its distribution affect the molecule's interaction with the electric field.

6. Shape of the Macromolecule:

   • The shape influences how easily the molecule can move through the matrix.

Laws Governing Electrophoresis

Electrophoresis is governed by electrostatic laws:

1. First Law: The movement of an ion with charge $q$ in an electric field is determined by the electrical force, $F<em>{\text{electric}}$, expressed as: $F</em>{\text{electric}} = qE$, where $E$ is the strength of the electric field.

2. Second Law: The movement of an ion in an electric field is opposed by a frictional force, $F<em>{\text{friction}}$, expressed as: $F</em>{\text{friction}} = fV$, where $f$ is the frictional coefficient and $V$ is the rate of migration of the ion.

Constant Electric Field

• In a constant electric field, $F<em>{\text{electric}} = F</em>{\text{friction}}$.

• Thus, $qE = fV$.

Electrophoretic Mobility

• Electrophoretic mobility, $\mu$, is defined as the ratio of the velocity of the charged particle to the strength of the electric field: $\mu = \frac{V}{E} = \frac{q}{f}$.

• It represents the distance an ion moves in 1 second under the influence of an electric field of 1 V/cm. The unit is $cm^2 s^{-1} V^{-1}$.

• Particles migrate at a rate proportional to the strength of the electric field and inversely proportional to the frictional pull.

Extended Formula

• mobility can be extended as: $\mu = \frac{Ze}{6 \pi \eta R}$, where:

  • $Z$ = number of charges.

  • $e$ = electronic charge.

  • $\eta$ = solvent viscosity.

  • $R$ = particle radius.

  • $\pi = \frac{22}{7}$

Classification of Electrophoresis

There are two main classes:

1. Moving Boundary Electrophoresis:

   • Carried out in solution.

   • Largely outdated and mostly used in non-biological experiments.

2. Zone Electrophoresis:

   • Requires a solid or porous support.

   • Common supports include filter paper, cellulose acetate strips, and gels.

Advantages of Zone Electrophoresis

• Prevention of Convective Mixing: Convective mixing due to heat is prevented.

• Better Separation: Sharp or discrete bands are produced.

• Small Sample Volume: Requires only a small amount or volume of sample compared to moving boundary electrophoresis.

Types of Zone Electrophoresis

1. Paper Electrophoresis

2. Cellulose Acetate Electrophoresis

3. Capillary Electrophoresis

4. Gel Electrophoresis

Paper Electrophoresis

• Uses filter paper as a supporting medium.

• The paper has a slight adsorption capacity and uniform pore size.

• Suitable for separating small molecules like amino acids, proteins, hemoglobin, insulin, myosin, and albumin.

• The filter paper is moistened with a buffer, and the two ends are immersed in buffer reservoirs containing electrodes.

• The sample is applied at the center of the filter paper, and a direct current (100-150V) is applied.

• Ions migrate according to their charges.

• After electrophoresis, the electrophoretogram is dried, and the components are detected by staining.

Staining

• Ninhydrin: Detects amino acids, producing a purple color (yellow if proline is present).

• Coomassie Brilliant Blue or Ponceau S: Detects proteins.

• Ozone/Schiff Reagent: Used for lipoproteins; ozone reacts with double bonds of fatty acids, converting them to aldehydes, which then react with Schiff reagent to give a red color.

• Separated bands can be viewed under UV light.

Electrophoretogram Interpretation

• Negative ions move towards the positive electrode (anode).

• Positive ions move towards the negative electrode (cathode).

Cellulose Acetate Electrophoresis

• A modified version of paper electrophoresis.

• Uses cellulose acetate membrane filters instead of regular chromatography paper.

Capillary Electrophoresis (CE)

• A capillary tube is filled with a buffer at a specific pH.

• The sample is introduced into the capillary via pressure or electrokinetic injection.

• High voltage (>300 V/cm) is applied.

• Components migrate through the capillary at different speeds.

• Positive components migrate to the negative electrode, and negative components migrate to the positive electrode.

• A detector assesses the rate of migration of the separated components.

• The best pH for separation is usually between the pKa values of the sample components.

• Capillaries are often made of bare fused silica, which has silanol groups (Si-O-H) on its surface, making it slightly acidic.

Types/Modes of Capillary Electrophoresis

1. Capillary Gel Electrophoresis (CGE)

2. Micellar Electrokinetic Capillary Chromatography (MEKC)

3. Capillary Electrochromatography (CEC)

4. Capillary Isoelectric Focusing (cIEF)

5. Capillary Isotachophoresis (CITP)

System Types

• Continuous System: Uses a background electrolyte throughout the capillary.

• Discontinuous System: Keeps the sample in distinct zones separated by different electrolytes (e.g., Sodium Dodecyl Sulfate - SDS).

Gel Electrophoresis

• Includes Polyacrylamide Gel Electrophoresis (PAGE), SDS-PAGE, and Agarose Gel Electrophoresis.

• Used for separating peptides, proteins, lipoproteins, DNA, and RNA.

• Suppresses thermal convection caused by the electric field and acts as a molecular sieve.

• Smaller molecules migrate faster than larger molecules in electrophoretic mobility.

Common Gel Types

• Polyacrylamide gels and agarose gels with specific pore sizes are commonly used.

Separation Basis

• Based on electrophoretic mobility and gel filtration.

• Smaller molecules migrate farther than larger ones, unlike gel filtration.

Polyacrylamide Gel Electrophoresis (PAGE)

• Based on electrophoretic mobility and charge.

• The gel is prepared from:

  1. Tetramethylethylene Diamine (TEMED)

  2. Ammonium Persulphate

  3. Acrylamide

  4. Bis-acrylamide

  Reagents

  • Ammonium Persulphate: Strong oxidizing agent and free radical initiator, converts TEMED to a radical.

  • TEMED: Converts acrylamide to a radical, initiating polymerization of the monomer.

  • Bis-acrylamide: Forms cross-links between acrylamide monomers.

• TEMED and ammonium persulphate are catalysts for the polymerization of acrylamide and bis-acrylamide gels.

• The four substances are dissolved in Tris buffer (pH 8 or 8.3) and cast into a gel cast plate (rod or rectangular shape).

• The same buffer is used to run the electrophoresis, imparting a net negative charge to the molecules, causing them to migrate to the anode.

• A direct current of ~300V is applied, and the current is allowed to pass through the gel for ~5 hours to separate the components into discrete bands.

• A tracking or loading dye (glycerol for density and bromophenol for color) is added to the sample.

• After electrophoresis, the gel is stained with Coomassie Brilliant Blue or Ponceau S or visualized using UV light.

• Amido Black, Bis-1-Anilino-8-Naphthalene Sulphonate, and Nile Red can also be used for staining.

Applications

• PAGE is commonly used to determine the molecular weights of proteins and the number of proteins in a mixture.

SDS-PAGE

• Sodium Dodecyl Sulphate (SDS) is added to the polyacrylamide gel.

• SDS confers a large negative charge on proteins, masking their individual charges.

• Allows separation on the basis of molecular weights with high accuracy.

• Disrupts non-covalent interactions between subunits, enabling the determination of subunit molecular weights.

• Proteins of known molecular weights (standards) are run alongside the sample.

Agarose Gel Electrophoresis

• Used for separating nucleic acids (DNA and RNA).

• Agarose gel provides large pore sizes suitable for separating nucleic acids.

• Agarose powder is dissolved in TBE or TAE buffer to form a slurry.

  Buffers

  • TBE Buffer: Tris-Borate-EDTA, prepared from Tris base, boric acid, and EDTA.

  • TAE Buffer: Tris-Acetate-EDTA, prepared from Tris base, acetic acid, and EDTA.

• The agarose slurry is heated until it melts into a homogenous solution, then cooled to ~45°C and poured into a gel caster or plate with a comb to create sample wells.

• The sample is loaded into the wells with a tracking dye composed of glycerol, bromophenol blue, and xylene cyanol.

• After electrophoresis, the gel is stained with ethidium bromide (5 μg/ml) and visualized under UV light to reveal nucleic acid bands.

• Silver chloride may also be used to stain DNA.

• Ethidium bromide intercalates within the nitrogenous bases of nucleic acids.

• Kilobase DNA ladder is loaded in the first and last lanes as a marker.

• A negative control sample is used, and a separate lane contains the actual sample.

Isoelectric Focusing (IEF)

• Also known as electro-focusing.

• Peptides and proteins are separated based on their isoelectric points (pI) in an electric field.

Isoelectric Point (pI)

• The pH at which the positive and negative charges on a molecule cancel out to zero.

• At a pH below the pI, the molecule is positively charged, and above the pI, it is negatively charged.

• Molecules remain in their specific isoelectric points, forming sharp and distinct bands.

• A pH gradient is applied to the gel during preparation, made using organic acids and bases that have both acidic and basic groups.

Applications of Gel Electrophoresis

1. Verify or assess the level of gene amplification by PCR or other sequencing reactions.

2. Assess the quality and quantity of genomic DNA after DNA extraction.

3. Separate DNA fragments after cleavage with endonucleases.

4. Visualize bands of a molecular marker to genotype individual plants.

5. Investigate various binding modes of small molecules to proteins.

6. Determine supercoiled DNA.

7. Determine the number of subunits in a protein.

Assignment

1. Southern blotting

2. Western blotting

Blotting involves the transfer of biological samples from a gel to a membrane, followed by their subsequent detection on the surface of the membrane.

Southern Blotting

• A method used in molecular biology for the detection. (The context ends abruptly here.)

Southern Blotting

- A method used in molecular biology for the detection of a specific DNA sequence in DNA samples.

1. DNA Extraction and Digestion:
   • The DNA is extracted from a sample and digested into fragments using restriction enzymes.
2. Gel Electrophoresis:
   • The DNA fragments are separated by size using agarose gel electrophoresis.
3. DNA Denaturation:
   • The DNA within the gel is denatured into single strands using an alkaline solution (e.g., NaOH).
4. Blotting:
   • The single-stranded DNA is transferred from the gel onto a membrane (usually nitrocellulose or nylon).
   • This can be done by capillary action, vacuum blotting, or electroblotting.
5. Hybridization:
   • The membrane is incubated with a labeled probe (a single-stranded DNA fragment complementary to the sequence of interest).
   • The probe is labeled with a radioactive isotope or a fluorescent marker.
6. Washing:
   • Excess or unbound probe is washed away.
7. Detection:
   • The hybridized probe is detected using:
     • Autoradiography (if the probe is radioactively labeled).
     • Fluorescence imaging (if the probe is fluorescently labeled).

Western Blotting

• A technique used to detect specific proteins in a sample.

1. Sample Preparation:
   • The sample is prepared by lysing cells or tissues and extracting the proteins.
   • Proteins are denatured and given a negative charge by treating them with sodium dodecyl sulfate (SDS).
2. Gel Electrophoresis:
   • The proteins are separated by size using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).
3. Blotting:
   • The separated proteins are transferred from the gel onto a membrane (usually nitrocellulose or PVDF).
   • This is typically done by electroblotting.
4. Blocking:
   • The membrane is blocked with a protein-rich solution (e.g., milk or BSA) to prevent non-specific binding of antibodies.
5. Primary Antibody Incubation:
   • The membrane is incubated with a primary antibody that specifically binds to the protein of interest.
6. Washing:
   • Excess or unbound primary antibody is washed away.
7. Secondary Antibody Incubation:
   • The membrane is incubated with a secondary antibody that binds to the primary antibody.
   • The secondary antibody is conjugated to an enzyme (e.g., horseradish peroxidase or alkaline phosphatase) or a fluorescent marker.
8. Washing:
   • Excess or unbound secondary antibody is washed away.
9. Detection:
   • The presence of the protein is detected using a substrate that reacts with the enzyme conjugated to the secondary antibody, producing a visible signal.
     • For HRP, chemiluminescence is commonly used.
     • For alkaline phosphatase, a chromogenic substrate can be used.
   • Alternatively, if the secondary antibody is fluorescently labeled, fluorescence imaging is used for detection.

Differences Between Southern and Western Blotting

1. Target Molecule:
   • Southern blotting detects DNA.
   • Western blotting detects proteins.