Electrophoresis

Module 5: Electrophoresis

1. Principles of Electrophoresis

Electrophoresis is a technique used to separate charged molecules, such as proteins, nucleic acids, and small ions, under the influence of an electric field. The molecules move through a gel or liquid medium at a rate that depends on their charge, size, and shape. This method is fundamental in molecular biology, biochemistry, and genetics for analyzing and purifying biomolecules.

2. Types of Electrophoresis

A. Native Polyacrylamide Gel Electrophoresis (Native PAGE)

Principle:
- Native PAGE separates proteins or nucleic acids based on their charge-to-mass ratio without denaturing them. The proteins or nucleic acids maintain their natural conformation and biological activity during the separation process. The separation is influenced by the charge, size, and shape of the molecules.
Procedure:
1. Gel Preparation:
  - Prepare a polyacrylamide gel with the desired concentration (e.g., 4-20%) based on the size of the proteins to be separated.
  - Cast the gel between two glass plates and allow it to polymerize.
2. Sample Preparation:
  - Mix the protein or nucleic acid samples with a loading buffer that does not denature the molecules.
  - Load the samples into the wells of the gel.
3. Electrophoresis:
  - Fill the electrophoresis tank with a running buffer that maintains the pH and ionic strength.
  - Place the gel in the tank and apply a voltage (e.g., 100-200V).
  - Run the gel until the tracking dye reaches the bottom of the gel.
4. Staining and Visualization:
  - Stain the gel with a protein stain (e.g., Coomassie Blue) to visualize the separated proteins.
  - De-stain to remove the background stain and enhance the visibility of the bands.
Applications:
- Protein Complex Analysis: Studying protein-protein interactions without disrupting native conformation.
- Conformational Studies: Observing conformational changes in proteins due to various factors.
- Enzyme Activity Assays: Directly assessing the activity of enzymes after separation.
Advantages:
- Preserves protein structure and function.
- Allows the study of protein complexes and interactions.
- Suitable for enzyme assays.
Disadvantages:
- Lower resolution compared to denaturing gels like SDS-PAGE.
- Cannot be used to determine the molecular weight of proteins precisely.

B. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Principle:
- SDS-PAGE separates proteins based on their molecular weight. The technique involves denaturing proteins with SDS, which binds uniformly to the polypeptides, imparting a negative charge proportional to their length. This uniform charge allows separation based solely on size as the proteins migrate through a polyacrylamide gel under an electric field.
Procedure:
1. Gel Preparation:
  - Prepare the stacking and separating gels with appropriate acrylamide concentrations (e.g., 4% for stacking gel, 10-15% for separating gel).
  - Pour the separating gel between glass plates and allow it to polymerize, then add the stacking gel on top.
2. Sample Preparation:
  - Mix protein samples with SDS sample buffer containing SDS, reducing agents (e.g., β-mercaptoethanol), and a tracking dye.
  - Heat the samples at 95°C for 5 minutes to denature the proteins.
3. Electrophoresis:
  - Load the denatured protein samples into the wells of the gel.
  - Run the gel at a constant voltage (e.g., 100-200V) until the tracking dye reaches the bottom.
4. Staining and Visualization:
  - Stain the gel with Coomassie Blue or silver stain to visualize the protein bands.
  - De-stain to remove the background and enhance the visibility of the bands.
Applications:
- Protein Molecular Weight Determination: Estimating the molecular weight of proteins by comparing them to a standard.
- Protein Purity Analysis: Assessing the purity of protein samples.
- Western Blotting: Transferring proteins to a membrane for antibody detection.
Advantages:
- High resolution for separating proteins based on size.
- Suitable for a wide range of molecular weights.
- Can be used to estimate protein molecular weight.
Disadvantages:
- Denatures proteins, preventing the analysis of native structures and functions.
- Requires careful handling of hazardous chemicals like acrylamide.

C. Agarose Gel Electrophoresis

Principle:
- Agarose gel electrophoresis is used to separate nucleic acids (DNA and RNA) based on size. The negatively charged nucleic acids migrate through the agarose gel towards the positive electrode, with smaller fragments moving faster and further through the gel.
Procedure:
1. Gel Preparation:
  - Dissolve agarose in buffer (e.g., TBE or TAE) by heating until fully melted.
  - Pour the molten agarose into a gel tray with a comb to create wells and allow it to solidify.
2. Sample Preparation:
  - Mix DNA or RNA samples with a loading buffer containing a tracking dye.
  - Load the samples into the wells of the gel.
3. Electrophoresis:
  - Submerge the gel in an electrophoresis tank filled with the same buffer used to prepare the gel.
  - Apply a constant voltage (e.g., 80-120V) until the tracking dye has migrated sufficiently.
4. Staining and Visualization:
  - Stain the gel with a nucleic acid stain (e.g., ethidium bromide, SYBR Green) to visualize the DNA or RNA bands under UV light.
Applications:
- DNA Fragment Analysis: Separating DNA fragments generated by restriction enzymes or PCR.
- RNA Analysis: Checking RNA integrity or analyzing RNA transcripts.
- DNA Fingerprinting: Comparing DNA samples for forensic analysis.
Advantages:
- Easy to prepare and handle.
- Suitable for separating large DNA or RNA fragments.
- Non-toxic in solid form.
Disadvantages:
- Lower resolution compared to polyacrylamide gels.
- Limited to nucleic acid separation.

D. Capillary Electrophoresis (CE)

Principle:
- Capillary Electrophoresis (CE) separates ions based on their size and charge within a narrow capillary filled with an electrolyte. The electric field drives the migration of ions through the capillary, with their migration velocity determined by their charge-to-mass ratio.
Procedure:
1. Capillary Preparation:
  - Select a fused silica capillary with appropriate dimensions (e.g., 50-100 µm internal diameter).
  - Rinse the capillary with a conditioning solution to remove any contaminants.
2. Sample Injection:
  - Inject the sample into the capillary using either hydrodynamic or electrokinetic injection methods.
3. Electrophoresis:
  - Apply a high voltage (e.g., ±30 kV) across the capillary to initiate the separation.
  - Monitor the separation process using a detector (e.g., UV, fluorescence).
4. Data Analysis:
  - Analyze the resulting electropherogram, which plots the detector response as a function of migration time.
Applications:
- Small Molecule Analysis: Separation and analysis of small ions, amino acids, and peptides.
- Pharmaceutical Testing: Assessing the purity and concentration of drugs.
- Forensic Science: DNA analysis in forensic investigations.
Advantages:
- High resolution and sensitivity.
- Requires very small sample volumes.
- Fast separation times.
Disadvantages:
- Requires specialized equipment.
- Capillaries can become clogged or damaged.
- Not suitable for very large molecules.

E. Isoelectric Focusing (IEF)

Principle:
- Isoelectric Focusing (IEF) separates proteins based on their isoelectric point (pI). Proteins migrate through a pH gradient in the gel until they reach the pH that matches their pI, where they have no net charge and stop moving.
Procedure:
1. Gel Preparation:
  - Prepare a polyacrylamide gel containing carrier ampholytes to establish a pH gradient.
  - Cast the gel and allow it to polymerize.
2. Sample Preparation:
  - Mix protein samples with a sample buffer and apply them to the gel.
3. Electrophoresis:
  - Apply a high voltage (e.g., 1000-2000V) to the gel to initiate the focusing of proteins along the pH gradient.
  - Run the gel until the proteins have reached their isoelectric points and stopped migrating.
4. Staining and Visualization:
  - Stain the gel to visualize the focused protein bands.
  - Analyze the pI values based on the position of the bands in the gradient.
Applications:
- Protein Purification: Separating proteins with very similar molecular weights but different pI values.
- Post-Translational Modifications: Detecting modifications that alter the pI of proteins.
- Proteomics: Used in two-dimensional electrophoresis for comprehensive protein analysis.
Advantages:
- High resolution for separating proteins with small differences in pI.
- Useful for analyzing protein modifications.
- Compatible with two-dimensional gel electrophoresis.
Disadvantages:
- Complex gel preparation and handling.
- Requires precise control of pH gradients.
- Not suitable for very basic or very acidic proteins.

F. Pulsed Field Gel Electrophoresis (PFGE)

Principle:
- Pulsed Field Gel Electrophoresis (PFGE) separates large DNA molecules by periodically changing the direction of the electric field, allowing large DNA fragments to reorient and migrate through the gel at different rates based on size.
Procedure:
1. Gel Preparation:
  - Prepare an agarose gel with a low concentration (e.g., 1%) to create large pores suitable for large DNA fragments.
  - Cast the gel in a mold and allow it to solidify.
2. Sample Preparation:
  - Embed DNA samples in agarose plugs to protect them during the lysis and electrophoresis processes.
  - Treat the plugs with enzymes or chemicals to release the DNA fragments.
3. Electrophoresis:
  - Place the gel in a PFGE chamber with electrodes arranged to switch the electric field direction.
  - Apply the pulsed electric field, typically at low voltage (e.g., 6-10V/cm) for extended periods (e.g., 24-48 hours).
4. Staining and Visualization:
  - Stain the gel with ethidium bromide or a similar DNA stain.
  - Visualize the DNA bands under UV light and analyze the migration pattern.
Applications:
- Epidemiological Studies: Genotyping and subtyping bacterial pathogens to track outbreaks.
- Genome Mapping: Analyzing large DNA fragments for physical mapping of genomes.
- Pathogen Identification: Differentiating bacterial strains in clinical and food microbiology.
Advantages:
- Capable of separating very large DNA molecules (up to several megabases).
- High resolution for analyzing complex genomes.
- Widely used in epidemiological studies.
Disadvantages:
- Time-consuming (can take several days).
- Requires specialized and expensive equipment.
- Limited to large DNA fragments and not suitable for small DNA molecules.

3. Supporting Media Used in Electrophoresis

Polyacrylamide Gels: Polyacrylamide gels are versatile and widely used in electrophoresis, particularly for protein separation (in SDS-PAGE, Native PAGE, and IEF). The concentration of acrylamide determines the pore size, which is critical for resolving molecules of different sizes. Polyacrylamide is chemically inert, electrically neutral, and hydrophilic, making it an ideal medium for electrophoresis.
Agarose Gels: Agarose is the medium of choice for nucleic acid separation in agarose gel electrophoresis and PFGE. Its large pore size is suitable for separating DNA and RNA molecules, particularly large fragments. Agarose gels are easy to prepare, and the concentration of agarose can be adjusted to create gels with different pore sizes, allowing for the separation of a wide range of nucleic acid sizes.
Capillary Tubes: In Capillary Electrophoresis, narrow capillary tubes filled with an electrolyte solution serve as the separation medium. The small diameter of the capillary allows for high-resolution separation and efficient heat dissipation, which is essential for maintaining sharp peaks during analysis.

Conclusion

Electrophoresis is a powerful and versatile technique used across various scientific fields to separate, analyze, and purify molecules based on their size, charge, and other properties. Understanding the principles, procedures, applications, advantages, and disadvantages for each type of electrophoresis—Native PAGE, SDS-PAGE, agarose gel electrophoresis, capillary electrophoresis, isoelectric focusing, and pulsed field gel electrophoresis—provides a comprehensive foundation for effectively utilizing these techniques in research and diagnostics. Each method has its unique strengths, making it suitable for specific types of analyses and applications, from protein purification and nucleic acid analysis to pathogen identification and genome mapping.

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Krishna Teja