Gel Electrophoresis | Zoology Optional Notes for UPSC PDF Download

Principle of Gel Electrophoresis

Upon subjecting these charged biomolecules to an electric field, they exhibit distinct migration patterns. This behavior arises from variations in their mass and net charge. The negatively charged particles, exemplified by nucleic acids, migrate towards the anode, while positively charged ones move in the direction of the cathode. The migration patterns depend on each particle's unique properties, including speed and direction alterations, ultimately allowing for the separation of biomolecular components with similar characteristics.

Components of the Gel Electrophoresis Apparatus

Power Supply

• Electrophoresis conditions: The apparatus operates under constant current, voltage, or power.
• Steady power supply: A stable power source is essential to maintain consistent migration rates.
• Lead cables: Red (anode/positively charged electrode) and black (cathode/negatively charged electrode) cables connect the power supply to the gel box.
• Current delivery: These cables transport the electric current from the power source to the gel box.
• Heat generation: An increase in current can lead to greater heat production due to resistance, causing thermal stirring of dissolved ions and quicker water evaporation.
• Ion concentration: The rising heat can elevate ion concentration in the buffer.
• Charge-based separation: DNA and RNA, being negatively charged, migrate from the cathode (black wire end) to the anode (red wire end).

Buffers

• pH control: Buffers are responsible for setting the system's pH and establishing electrical charges on the solute.
• Ideal buffer properties: A suitable buffer should maintain the analyte's solubility, consistent buffering capacity throughout the analysis, not interfere with target analyte detection, and achieve the required separation range.
• Acidic and Basic buffers: Acidic buffers like citrate, acetate, formate, and phosphate are employed for lower pH, while basic buffers such as tric, borate, and tricine maintain higher pH levels.
• Ionic composition: Buffers are composed of monovalent ions with equivalent valency and molality.
• Buffer temperature: Preparing buffers in advance, chilling them, and using cold buffers during the procedure can enhance sample resolution, reduce solvent evaporation, and minimize bacterial growth.
• Buffer reusability: Large volumes of buffer can be reused up to four times, although smaller volumes may be discarded. High ionic strength of the buffer can offer sharper resolution but poses a risk to heat-labile chemicals due to the high heat generated.

Support Media

• Starch, Polyacrylamide, Agarose, and Cellulose Acetate: These materials are utilized as supporting media in the form of sheets, slabs, and columns.
• Colloidal nature: They consist of over 90% water and function as molecular sieves to separate molecules.
• Pore size selectivity: Their porous nature allows small molecules to pass while retaining larger molecules.
• Electrical neutrality: Ensuring electrical neutrality is essential.
• Agarose preference: Agarose gel is commonly used as a support medium during electrophoresis.

Starch Gel

• Early gel medium: Historically, starch gel was the first medium used for electrophoresis.
• Separation based on properties: It enabled the separation of proteins according to their charge-to-mass ratio and molecular size.
• Colloidal suspension: A colloidal suspension is formed by boiling starch granules in a buffer, resulting in a semi-solid gel as it cools.
• Use of petroleum jelly: To prevent swelling and shrinking, petroleum jelly is added.
• High resolving power: This method can achieve sharp zones and high resolving power, but its lack of reproducibility limits its current usage.

Cellulose Acetate

• Hemoglobin separation: Cellulose acetate electrophoresis was initially developed to separate hemoglobin in red blood cells and detect abnormal hemoglobin in blood serum.
• Acetylated filter papers: Cellulose is acetylated to produce cellulose acetate, with acetylation mainly occurring at the glucose ring's C-3 and C-6 positions.
• Pore size advantage: Compared to other electrophoretic matrices like agarose and polyacrylamide, cellulose acetate has larger pores.

Agarose

• Natural linear polymer: Agarose, derived from red seaweeds, is a linear polymer composed of galactose and 3,6-anhydro-galactose chains.
• Dry powder storage: Agarose is typically stored in a dry powder form.
• Gel casting: It is cast by dissolving the agarose powder in an appropriate solution buffer, heating it, and allowing it to cool to room temperature.
• Pore size control: The agarose concentration in the solution buffer determines the gel's pore size.
• DNA and RNA separation: Agarose gel is frequently used to distinguish between DNA and RNA molecules, typically at concentrations ranging from 0.8% (W/V) to 5% (W/V).
• Resolution compared to polyacrylamide gels: Agarose gels offer relatively lower resolution but are favored for their low gelling temperature, neutral charge, and stable gel formation, making them suitable for gel electrophoresis in various forms, either solid or liquid.

Polyacrylamide

• Transparent gel: Polyacrylamide gels are clear and transparent, formed through the copolymerization of acrylamide monomers in the presence of the crosslinking agent N,N-methylene-bis-acrylamide (also known as "bis-acrylamide").
• Pore size control: The acrylamide concentration, proportional to its crosslinking agent, determines pore size in polyacrylamide gels.
• DNA and protein separation: These gels are employed for both DNA and protein separation, with lower acrylamide concentrations (3%-15%) used for DNA and higher percentages (10%-20%) for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins under denatured conditions based on their size.

Electrophoresis Chamber

• Buffer-filled container: The electrophoresis chamber is a plastic tank filled with buffer, preventing the movement of biomolecules.
• Transparent lid: A transparent lid facilitates visual observation of the migration process.
• Wired to power supply: The chamber is wired to a power supply to create an electric field.

Container for Staining and De-Staining Gel

• Tray and container use: Staining and de-staining of gels are achieved using trays and containers.
• Lidded and open-form options: These containers come in lidded and open-form versions.
• Propylene base: They typically feature a propylene base.
• Chemical resistance: These containers are resistant to chemicals and staining.

Electrodes

• Platinum electrodes: The apparatus includes two platinum electrodes responsible for separating molecules due to their ability to attract opposite charges.
• Anode and cathode: An anode binds positive ions, while a cathode binds negative ions.

Gel Caster and Comb

• Gel casting: The gel is poured into a gel caster, which contains the gel and is stored within the apparatus once it has dissolved in the solvent.
• Well preparation: A comb is used to prepare wells for sample loading.

These components collectively form the essential apparatus for conducting gel electrophoresis experiments.

Types of Electrophoresis

1. Paper Gel Electrophoresis:

   • Used for investigating serum and bodily fluids in clinical settings.
   • Non-transparent and non-toxic.
   • Convenient for storage.
   • Prone to protein adsorption, poor conductivity, and background staining.
   • Cellulose's OH groups can slow electrophoretic motion and reduce resolution.

2. Agarose Gel Electrophoresis:

   • The concentration of agarose determines resolution.
   • Suitable for separating DNA fragments (100 base pairs to 20 kilobase pairs) and proteins.
   • Low-concentration agarose can perform isoelectric focusing for amphoteric molecules.

3. Polyacrylamide Gel Electrophoresis (PAGE):

   • Used at varying concentrations (3-30%) depending on the application (proteins or DNA).
   • Offers high reliability, accurate porosity, and is used for various applications, including DNA sequencing and RNA separation.

4. Pulse-Field Gel Electrophoresis (PFGE):

   • Introduced in 1984.
   • Used to separate high molecular weight DNA, including entire chromosomes, by altering the electric field's direction and strength.
   • Provides precise and reproducible results for various applications.

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

   • Proteins are separated based on polypeptide chain length.
   • SDS and polyacrylamide gel disrupt the structure and charge of proteins.
   • Used to determine protein molecular weight and sample purity.

6. 2D Electrophoresis:

   • Analyzes complex protein mixtures by combining isoelectric focusing (IEF) and SDS-PAGE.
   • Proteins are separated by charge (IEF) and then by mass (SDS-PAGE).
   • Effective for resolving complex protein mixtures.

7. Immuno-Electrophoresis (Rocket Electrophoresis):

   • Combines electrophoresis and immunodiffusion.
   • Used to separate protein antigens in semi-solid media and create precipitin arcs.
   • Suitable for measuring antigens using antibodies in agarose gels.

8. Difference Gel Electrophoresis (DIGE):

   • Developed for quantitative differential-expression studies.
   • Up to three different protein samples are labeled with fluorescent dyes.
   • Samples are combined, loaded, and subjected to 2D electrophoresis for quantitative analysis.

These different types of electrophoresis techniques have specific applications and advantages, making them valuable tools in various fields of research, diagnostics, and molecular biology. They are categorized based on the nature of the molecules being separated (e.g., DNA, RNA, proteins) and the specific goals of the experiment (e.g., quantitative analysis, high-resolution separation).

Gel Electrophoresis Operating Procedures

• Initiate by dissolving the gel in boiling water, followed by cooling it to a suitable temperature.
• Pour the gel solution into a casting mold.

2. Gel Casting and Well Formation

• Once the gel has solidified, employ a comb to create wells within it.
• Place the prepared gel into the electrophoretic chamber, ensuring that the buffer level does not exceed one-third of the chamber's volume.

3. Sample Preparation

• Enhance the sample's visibility and density by introducing loading dye, which may be a fluorescent tag or ethidium bromide.
• Isolate and pre-process the DNA, incorporating a basic blue dye to facilitate monitoring the sample's migration within the gel.

4. Sample Loading

• Load the sample into the wells using a clean micropipette.

5. Electrophoresis

• Connect the electrophoretic chamber to a power supply, with the negative and positive leads properly attached.
• Upon activating the power supply, an electric field is established, generating negatively charged particles. Consequently, negatively charged DNA molecules migrate towards the anode, while other molecules move in the opposite direction.

6. Stopping Electrophoresis, Staining, and Visualization

• Cease electrophoresis by deactivating the power supply.
• Visualize the sample migration by employing a suitable dye.
• After the procedure, stain and visualize the gel using a gel imager.
• Determine the sizes of sample fragments by comparing them to a standard, utilizing the logarithm of molecular weight for accurate size estimation.

Limitations of Electrophoresis

1. Limited Sample Analysis

• In contrast to methods like in situ hybridization (ISH), electrophoresis can only analyze a limited number of regions within a tissue sample.

2. Precision

• While gel electrophoresis effectively separates proteins, mass spectrometry is needed for precise mass determination.

3. Substantial Starting Sample

• Proteins cannot be efficiently amplified like DNA and RNA, necessitating larger tissue samples for analysis.

4. Limited Visualization

• Ineffective for measuring small molecules such as hormones and ions due to their size and reactivity issues.

5. Low Throughput

• Provides data relatively slowly compared to other high-throughput methods like PCR and flow cytometry.

Precautions for Electrophoresis

• Use nonconducting floors and benches, made of materials like wood or plastics, to avoid electrical interference.
• Avoid contact with unintended grounding points and conductors, such as sinks and waste sources, when working with electrophoresis systems.
• Handle samples with care while loading them into wells to prevent well damage.
• Wear gloves, face masks, and goggles when preparing gels, especially when handling hazardous substances like Ethidium Bromide (EtBr), which is carcinogenic and mutagenic. Take appropriate safety precautions.