3. Electrophoresis


Introduction to Electrophoresis

  • Electrophoresis: The migration of charged molecules in solution in response to an electric field.

  • Gel Electrophoresis: A specific type of electrophoresis where charged molecules migrate through a gel under the influence of an electric current.

Laboratory Safety in Electrophoresis

  • High risk of electric shock.

  • Precautions include:

    • Use electrophoresis equipment with electrical interlocks to interrupt current flow when the box is opened.

    • Regularly verify the integrity of the electrophoresis tank to detect leaks or damage.

    • Position electrophoresis apparatus away from high-traffic areas and provide physical barriers to discourage accidental contact.

    • Display proper warnings about high voltage.

Purpose of Electrophoresis

  • Designed to separate biological macromolecules such as DNA, RNA, or proteins.

  • The output results in distinct bands, each representing individual proteins or DNA fragments (e.g., immunoglobulins, albumin).

Gel Composition and Types

Gel Description

  • Gel: A jello-like matrix with pores through which biological molecules migrate.

  • Composed of acrylamide or agarose.

Agarose Gels

  • Definition: Made from seaweed; consists of a chain of sugar molecules.

  • Properties:

    • Comes in powdered form and is soluble in boiling water.

    • Forms a firm gel when cooled to room temperature.

    • The density or pore size is determined by the amount of agarose used.

  • Applications:

    • Estimating DNA fragment sizes for restriction mapping.

    • Analyzing PCR products.

    • Estimating DNA concentrations.

    • Examining integrity of DNA/RNA samples.

    • Purifying fragments for subsequent analyses.

    • Screening proteins for abnormalities.

Polyacrylamide Gels

  • Definition: Made from a mixture of acrylamide and bis-acrylamide.

  • Properties:

    • Polymerizes to form a network (weave) requiring catalyst (APS) and stabilizer (TEMED).

    • Bis-acrylamide increases weave density.

    • It's challenging to polymerize when exposed to oxygen.

  • Two types:

    • Non-denaturing (29:1):

    • Used to separate small double-stranded DNA fragments.

    • Denaturing (19:1):

    • Used to separate single-stranded DNA, which falls apart (non-functional).

  • Applications:

    • High-resolution fragment separation for sequencing.

    • Analysis of PCR products.

    • Purifying fragments.

    • Screening proteins for abnormalities.

    • Specific techniques: SDS-PAGE, 2D SDS-PAGE, and BN-PAGE.

Comparing Agarose and Polyacrylamide Gels

Agarose

  • Easy to prepare and non-toxic.

  • Lower resolution compared to polyacrylamide.

  • Operates at lower voltage (approximately 100V) and melts with increased heat.

Polyacrylamide

  • Higher resolution, can load larger quantities, operates at higher voltages (approximately 300V).

  • Acrylamide is a neurotoxin and must be handled carefully.

Gel Apparatus Components

  • Electrophoretic tank.

  • Buffer solution.

  • Power supply.

Running a Gel

  • Methods involve casting and running either agarose or polyacrylamide gels.

Principles of Gel Electrophoresis

Power and Resistance

  • Molecules move within a gel when an electric field is applied, with voltage driving the process.

  • Ohm’s Law: I=VRI = \frac{V}{R}, where I is current, V is voltage, and R is resistance.

Heat Production

  • Power: P=V×IP = V \times I signifies that more power results in more heat generated.

  • Power utilizing Ohm's law:
    P=I2×RP = I^2 \times R; this expresses the relationship between power, current, and resistance.

Electrophoresis Power Supply Settings

  • Constant Current:

    • Maintains current even as resistance increases.

    • Advantages: Constant migration rate and sharper bands.

    • Disadvantages: Increased heat over time and difficulty in running multiple gels.

  • Constant Voltage:

    • Keeps voltage steady while current and power decrease with resistance.

    • Advantages: Safer with less heat production, ability to run multiple chambers.

    • Disadvantages: Longer run times and more diffuse bands.

  • Constant Power:

    • Maintains power while current decreases with resistance.

    • Advantages: Constant heat production.

    • Disadvantages: Unpredictable migration rates, longer run times.

Factors Influencing Molecular Migration

General Factors

  • Separation is based on size and conformation of DNA and size, charge, and structure for proteins.

  • Migration rate is expressed as: αlog⁡10\alpha \log_{10}.

Specific Factors

  • Gel Concentration:

    • Higher gel concentration creates smaller pores, slowing larger fragments.

  • Ionic Strength of Buffer:

    • Higher ionic strength improves electric current but decreases electrophoretic mobility.

    • The gel must have matching ionic strength with the buffer to sustain current flow.

Buffer pH and Ionic Strength Effects

  • Inappropriate buffer pH can lead to improper migration.

  • Salt concentration impacts band quality.

  • High voltage can degrade gels and affect resolution.

Loading Dyes and Molecular Weight Markers

Loading Dyes

  • Functions:

    1. Provide visibility while the sample runs.

    2. Facilitate tracking progress.

    3. Ensure the sample sinks in wells.

  • Different dyes migrate at different rates.

  • Choosing the right dye is crucial to ensure it doesn’t interfere with the sample.

Molecular Weight Markers

  • Known size fragments, termed size ladders, help to estimate sample sizes within gels.

Gel Visualization Techniques

DNA/RNA Visualization

  • Ethidium Bromide (EtBr):

    • Intercalates into DNA and fluoresces under UV light, highly mutagenic.

  • SYBR Green and Safe:

    • Non-toxic alternatives more sensitive than EtBr.

Protein Visualization

  • Coomassie Stain:

    • Common dye for protein detection; available in G-250 and R-250 forms.

Specific Gel Electrophoresis Techniques

  • Several methods include Serum Protein Electrophoresis, Immuno-electrophoresis, SDS-PAGE, and Capillary Electrophoresis.

Serum Protein Electrophoresis

  • Diagnostic tool for various health conditions.

  • Band structure includes albumin and immunoglobulin components.

  • Function of albumin: Prevents fluid leakage and carries substances in blood.

Electroendosmosis

  • Refers to the movement of positive ions towards a negative electrode, potentially influencing the behavior of proteins during electrophoresis.

Immunoelectrophoresis and Immunofixation

  • Techniques that use antibodies for specific identification of antigens in the gel.

Conclusion and Applications

  • Applications of electrophoresis span across various fields, including diagnostics, research, and biotechnological developments.

  • Importance of parameters selection, including gel type and buffer conditions, for achieving accurate and reproducible results.