Electrophoresis is a key technique in molecular biology, biochemistry, and biotechnology utilized for the separation and analysis of macromolecules like DNA, RNA, and proteins based on attributes such as size and charge. This method operates on the principle that charged molecules will move when subjected to an electric field, enabling researchers to isolate complex mixtures into distinct components. Electrophoresis widely used in various fields, including genetic research, clinical diagnostics, forensic science, and protein analysis.

Principles of Electrophoresis

Electrophoresis operates on the principle of charged particles moving within an electric field. The main principles consist of:

• Charge and Mobility: Molecules like DNA, RNA, and proteins carry a net charge. Under an electric field, these molecules migrate toward the electrode with the opposite charge (for example, DNA, which has a negative charge, progresses toward the anode).
• Size and Shape: The speed of migration is influenced by the size and shape of the molecules. Smaller molecules move faster through the gel matrix, while larger molecules move more slowly.
• Gel Matrix: A porous gel (such as agarose or polyacrylamide) serves as a sieve, separating molecules according to their size. The size of the gel’s pores can be adjusted to enhance separation for specific molecules.
• Buffer System: A conductive buffer solution ensures a stable pH and supplies the necessary ions for the electric current to flow.
• History of Electrophoresis

The origins of electrophoresis date back to the 1930s when Arne Tiselius created the first apparatus for electrophoresis in 1937, enabling the separation of proteins within a U-shaped tube using an electric current. His pioneering efforts earned him the Nobel Prize in Chemistry in 1948 and established a basis for this technique. In the 1950s, the introduction of support matrices such as starch gels and cellulose acetate enhanced both the resolution and practicality of electrophoresis.

The 1960s saw the development of polyacrylamide gel electrophoresis (PAGE), particularly the SDS-PAGE method, which allowed for the separation of proteins based on their molecular weight. During the 1970s, agarose gel electrophoresis became prominent for analyzing DNA and RNA, together with techniques like Southern blotting. The 1980s introduced two-dimensional gel electrophoresis (2D-GE) aimed at proteomics research. The 1990s marked a significant shift with the advent of capillary electrophoresis (CE), which transformed high-throughput DNA sequencing. Currently, innovations in microfluidics, fluorescence detection, and automation are further broadening the applications of electrophoresis in both research and diagnostics.

Electrophoresis Equipment and Instrumentation

The basic setup for electrophoresis includes the following components:

1. Power Supply

• Creates a stable electric field for the movement of charged particles.
• Allows for voltage and current adjustments to accommodate various types of electrophoresis.
• Typical voltage ranges include:
  • Agarose gel electrophoresis: 50–150V
  • Polyacrylamide gel electrophoresis (PAGE): 100–300V

2. Gel Casting and Running Apparatus

• Gel Tray & Comb: Utilized to create gels with wells for sample insertion.
• Buffer Tank: Contains the buffer solution, providing ionic strength and stabilizing pH.
• Electrodes: Generally constructed from platinum or stainless steel, positioned at opposite ends of the tank.
• Lid with Safety Interlock: Prevents unintended exposure to electrical currents.

3. Gel Matrices

• Agarose Gel: Employed for the separation of DNA/RNA, formulated at varying concentrations (0.5–3%).
• Polyacrylamide Gel: Used for the separation of proteins and small DNA fragments, created using acrylamide and bisacrylamide.

4. Buffers

• Deliver ionic conductivity and help to sustain the pH of the system.
• Examples include:
  • TAE (Tris-Acetate-EDTA): Frequently used in agarose gel electrophoresis.
  • TBE (Tris-Borate-EDTA): Offers greater buffering capacity compared to TAE.
  • SDS (Sodium Dodecyl Sulfate): Applied in SDS-PAGE to denature proteins.

5. Sample Loading and Detection Tools

• Pipettes & Micropipettes: For accurate sample loading.
• Loading Dye: Enhances sample density and enables visualization during electrophoresis.
• Molecular Weight Markers (Ladders): Standard size references for comparison.

6. Staining and Visualization Equipment

• Ethidium Bromide (EtBr): A fluorescent dye for DNA that requires UV illumination.
• SYBR Green, GelRed: Alternative, safer stains for DNA.
• Coomassie Brilliant Blue, Silver Stain: Utilized for staining proteins in PAGE.
• UV Transilluminator or Gel Documentation System: Used to detect gels of fluorescently stained DNA/RNA.

7. Additional Equipment for Specific Techniques

• Capillary Electrophoresis (CE) System: Employs slender capillaries instead of gel for enhanced resolution in separation.
• Isoelectric Focusing (IEF) System: Separates proteins based on their isoelectric point (pI).
• 2D Gel Electrophoresis Equipment: Integrates IEF and SDS-PAGE for advanced protein analysis.

Gel Electrophoresis procedure:

Steps in Electrophoresis

• Step 1: Sample Preparation
  • Isolate the DNA and prepare the solution by adding blue dye (Ethidium Bromide (EtBr) so that it will be easy to observe the movement of the sample taking place in the gel.
• Step 2: Prepare an agarose TAE gel solution –
  • TAE buffer solution helps to generate an electric field during the process of electrophoresis. To prepare the solution, for example, if there is a requirement of 1% agarose gel then add 100mL TAE to 1 g of agarose. The higher percentage of agarose will give a denser screen. Dissolve the agarose by heating the agarose TAE solution.
• Step 3: Gel casting –
  • Pour the agarose TAE solution in a casting tray. Allow it to cool and solidify. A gel slab along with the wells is ready to use for the experiment.
• Step 4: How to set up the electrophoresis chamber?
  • Fill a chamber with TAE buffer. Place the solid gel in the chamber. Place the gel in such a position such that it is near the negative electrode.

• Step 5: Gel loading –
  • Load the wells with the DNA sample and DNA ladder (a reference for sizes).
• Step 6: Process of electrophoresis –
  • Connect the positive and negative points to the power supply and chamber. Switch on the power and migration in the DNA sample due to the electric field generated.
• Step 7: Expose the ethidium bromide stained gel under UV light
  •  After complete of processes switch off the power supply and examine the gel under UV light. The DNA bands appear in the lane of respective well. Also, the DNA ladder is visible.

How to Prepare Electrophoresis Buffer ?

To prepare an electrophoresis buffer, you can follow these general instructions for a typical running buffer such as TAE or TBE:

• Select the Buffer Type: Determine whether you will be using TAE (Tris-Acetate-EDTA) or TBE (Tris-Borate-EDTA).
• Gather Components:
• For TBE: Use the following measurements to create a 1 L solution of 10x TBE buffer:
  • 1 M Tris base: 121.14 g
  • 0.9 M Boric acid: 61.83 g
  • 0.02 M EDTA (disodium salt): 7.45 g
• For TAE: For a 1 L solution of 50x TAE buffer, utilize:
  • 2 M Tris base: 48.4 g
  • 1 M Acetic acid: 28.4 mL
  • 0.05 M EDTA: 7.45 g
• Dissolve and Adjust Volume: Mix the powders in approximately 500 mL of distilled or deionized water. Once completely dissolved, add distilled water until the total volume reaches 1 L.
• pH Verification: adjust the pH to 8.3, adjusting it with small quantities of concentrated HCl, acetic acid or NaOH.
• Storage: Keep the buffer at room temperature, shielded from light, and ensure it is labeled correctly.

Always double check and verify measurements in accordance with your experimental requirements and laboratory protocols.

Types of Support Media Used in Electrophoresis

Agarose Gel

Agarose gel, sourced from seaweed, consists of a polysaccharide matrix primarily used for the separation of large DNA and RNA fragments ranging from 0.1 to 50 kb. The pore size is influenced by the concentration of agarose (between 0.5% and 2%), where lower concentrations allow for the resolution of larger molecules. Agarose gels are economical, straightforward to create, and compatible with nucleic acid dyes such as ethidium bromide or SYBR Green. More advanced techniques like pulsed-field gel electrophoresis (PFGE) are employed to separate megabase-sized DNA through alternating electric fields. There is ongoing research on functionalized agarose gels that enhance the resolution and separation of intricate biomolecules, including glycoproteins and nucleic acid-protein complexes.

Polyacrylamide Gel

Polyacrylamide gel electrophoresis (PAGE) serves as the conventional method for the high-resolution separation of proteins and small nucleic acids. These gels are created by polymerizing acrylamide and bis-acrylamide, with the pore size regulated by the ratio of monomers to crosslinkers. SDS-PAGE denatures proteins to enable separation based on molecular weight, while native PAGE maintains the protein’s structure. Two-dimensional gel electrophoresis (2D-GE) integrates isoelectric focusing (IEF) and SDS-PAGE to facilitate proteomic analysis. Recent innovations include gradient gels that allow for the resolution of complex mixtures and fluorescent staining techniques that enhance sensitivity and quantification.

Capillary Electrophoresis (CE)

Capillary electrophoresis (CE) facilitates the separation of molecules within a narrow capillary containing a conductive buffer, providing high resolution and expedited analysis. Detection methods encompass UV-Vis, fluorescence, and mass spectrometry. CE is particularly well-suited for small molecules, peptides, and nucleic acids, while requiring minimal sample volumes. Recent developments involve the integration of CE with microfluidics and mass spectrometry for improved sensitivity, along with the use of coated capillaries to minimize analyte adsorption. Additionally, CE is being investigated for applications in single-cell analysis and point-of-care diagnostics.

Starch Gel

Starch gel, an early medium for electrophoresis, is utilized for the native separation of proteins, especially isoenzymes and allozymes in population genetics. Derived from hydrolyzed potato starch, it has fallen out of favor due to variability in gel quality and the advancement of polyacrylamide gels. Nonetheless, it continues to be valuable for analyzing historical samples and specialized applications. Recent research is investigating modified starch gels for uses in environmental and food science, including the detection of allergens or contaminants.

Cellulose Acetate

Cellulose acetate membranes facilitate quick protein separation in clinical diagnostics, specifically for identifying hemoglobin variants related to blood disorders like sickle cell anemia. These hydrophilic membranes are straightforward to handle and require small sample volumes. Recent innovations include biosensors based on cellulose acetate for point-of-care testing and their integration with microfluidic systems for streamlined analysis, making them viable options in resource-limited environments.

Paper Electrophoresis

Paper electrophoresis, one of the initial techniques developed, employs filter paper to separate charged molecules such as amino acids and small proteins. Although it has largely been supplanted by more advanced methods, it remains relevant for educational purposes and settings with limited resources. Current studies are examining chemically modified paper for targeted applications, including the separation of metal ions or the development of paper-based diagnostic tools.

Gradient Gels

Gradient gels, which feature varying concentrations of acrylamide, are effective at resolving molecules of different sizes within a single run. They excel in analyzing complex protein mixtures and identifying minor isoforms. Commonly employed in two-dimensional gel electrophoresis (2D-GE) for proteomics, they provide enhanced resolution for post-translational modifications and extracellular vesicles. The availability of pre-cast gradient gels enhances usability, while ongoing research focuses on refining formulations for particular uses.

Microfluidic Chips

Microfluidic chips condense the process of electrophoresis, allowing for the integration of sample preparation, separation, and detection into a single platform. They provide high resolution, rapid analysis, and require minimal sample sizes, which makes them particularly suitable for single-cell analysis and point-of-care diagnostics. Recent developments encompass lab-on-a-chip systems that combine electrophoresis with PCR or mass spectrometry for a thorough analysis. Their applications range from environmental monitoring and food safety to space research, where efficiency and portability are essential.

What are the Applications of Electrophoresis?

Electrophoresis is a flexible method with a wide range of applications:

• DNA Analysis:
  • Forensic DNA profiling.
  • Examination of PCR products.
  • Analysis of restriction fragment length polymorphism (RFLP).
• RNA Analysis:
  • Separation of RNA for Northern blot analysis.
  • Quality assessment of RNA samples.
• Protein Analysis:
  • Protein separation for Western blotting.
  • Evaluation of protein purity and size.
• Clinical Diagnostics:
  • Identification of genetic disorders.
  • Analysis of serum proteins for diagnosing diseases.
• Research:
  • Investigation of gene expression.
  • Research in proteomics and metabolomics.

Advancements in Electrophoresis

• Microchip Electrophoresis:
  • Compact systems that carry out electrophoresis on a microchip, minimizing the use of samples and reagents.
• Automated Systems:
  • High-capacity systems designed for extensive analysis, frequently utilized in genomics and proteomics.
• Fluorescence-Based Detection:
  • Improved sensitivity and specificity achieved through the use of fluorescent dyes and labels.
• Integration with Other Techniques:
  • Merging electrophoresis with mass spectrometry for enhanced protein analysis.