Overview of Two-Dimensional Gel Electrophoresis

What is 2D Gel Electrophoresis?

Two-dimensional gel electrophoresis (2DGE) is a powerful analytical technique used to separate complex mixtures of proteins based on two distinct properties: isoelectric point (pI) and molecular weight. This method provides a high-resolution approach to analyze the proteome, enabling the detailed study of protein expression, modification, and interaction in various biological samples.

Historical Background of 2D Gel Electrophoresis

Two-dimensional gel electrophoresis emerged as a revolutionary advancement in protein analysis in the 1970s. Initially, one-dimensional gel electrophoresis (1DGE) was the predominant method, separating proteins based solely on size. The introduction of 2DGE by O'Farrell in 1975 marked a significant leap, enabling the simultaneous separation of proteins based on both isoelectric point (pI) and molecular weight.

This dual separation approach addressed the limitations of 1DGE, which often failed to resolve complex protein mixtures with high precision. Since its inception, 2DGE has undergone various refinements, incorporating improvements in gel chemistry, staining methods, and image analysis techniques, thus solidifying its role in proteomics.

Principle of 2D Gel Electrophoresis

The principle of 2DGE is founded on the sequential application of two complementary electrophoretic techniques: isoelectric focusing (IEF) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Isoelectric Focusing (IEF)

The first dimension of 2DGE is IEF. This technique separates proteins based on their isoelectric point (pI), the pH at which a protein has no net electrical charge. The key principle of IEF is that proteins migrate in an electric field until they reach a region of the gel where the pH equals their pI, at which point they stop moving.

Mechanism:

• pH Gradient Creation: IEF involves the establishment of a pH gradient within a gel or on a membrane using ampholytes or other pH gradient systems. Ampholytes are molecules that distribute themselves throughout the gel to create a continuous gradient of pH values.
• Protein Focusing: When an electric field is applied, proteins move through the gradient until they reach their isoelectric point. At this point, the protein's net charge is neutralized, and it stops migrating. This results in a focusing effect where proteins with similar pI values group together in sharp, discrete bands.

SDS-PAGE (Second Dimension)

In the second dimension of 2DGE, SDS-PAGE separates proteins based on their molecular weight. Unlike IEF, which is based on charge, SDS-PAGE relies on the size of the proteins. SDS is a detergent that imparts a uniform negative charge to proteins, allowing them to be separated by size alone.

Mechanism:

• Denaturation and Uniform Charge: SDS binds to proteins, denaturing them and giving them a negative charge proportional to their length. This ensures that protein migration during SDS-PAGE is only influenced by their size, not their charge.
• Size-Based Separation: Proteins are then separated by size as they migrate through a polyacrylamide gel matrix. Smaller proteins move faster through the gel's porous network, while larger proteins move more slowly. This results in distinct bands of proteins with different molecular weights.

Integration of Dimensions

The integration of IEF and SDS-PAGE in 2DGE provides a comprehensive separation of proteins by combining charge-based and size-based resolution. By first resolving proteins according to their pI and then by their molecular weight, 2DGE offers high-resolution separation of complex protein mixtures.

Mechanism:

• Sequential Separation: Proteins are first focused in the IEF step, creating a high-resolution map of proteins based on their pI. The IEF gel is then subjected to SDS-PAGE, where proteins are further separated based on size. This two-step process results in a detailed two-dimensional separation of proteins.
• High-Resolution Profiling: The outcome is a 2D gel with each protein separated into distinct spots based on its unique combination of pI and molecular weight. This allows for the detailed analysis of complex proteomes, revealing both protein identity and abundance.

Procedure of 2D Gel Electrophoresis

Sample Preparation

Protein Extraction:

Cell or Tissue Lysis: Proteins are extracted from cells or tissues using a lysis buffer that typically contains detergents, salts, and protease inhibitors to prevent protein degradation. The buffer conditions should be optimized to solubilize a wide range of proteins while preserving their structure.

Clarification: The lysate is centrifuged to remove insoluble debris, leaving a clear supernatant containing the solubilized proteins.

Protein Quantification:

Quantitative Assays: The concentration of proteins in the extract is determined using assays such as the Bradford, BCA (bicinchoninic acid), or Lowry assay. Accurate quantification ensures that a consistent amount of protein is loaded onto the gel.

Sample Preparation for IEF:

Sample Dilution: The protein sample is diluted in an appropriate buffer, often containing ampholytes (which establish the pH gradient in the IEF gel) and other components to ensure optimal protein separation.

Reduction and Alkylation: Proteins may be reduced with agents like beta-mercaptoethanol or dithiothreitol to break disulfide bonds and alkylated with iodoacetamide to prevent reformation of disulfide bonds.

Isoelectric Focusing

Gel Preparation:

IEF Gel Casting: An IEF gel is prepared with a pH gradient. This gradient is typically created using ampholytes or other pH gradient systems incorporated into the gel matrix. The gel is cast between two glass plates to create a thin, uniform slab.

IEF Run:

Sample Application: Protein samples are applied to the gel using a sample applicator or a focusing cup. The sample is then focused along the pH gradient.

Electrophoresis: An electric field is applied, causing proteins to migrate through the gel until they reach their isoelectric point (pI), where their net charge is zero. This step may take several hours, depending on the size of the gel and the complexity of the sample.

Preparation for SDS-PAGE

Equilibration of IEF Gel:

Removal from IEF Setup: After focusing, the IEF gel is carefully removed from the focusing apparatus.

Equilibration: The IEF gel is equilibrated in a buffer containing SDS and a reducing agent. This step prepares the proteins for separation by SDS-PAGE. The SDS binds to the proteins, giving them a uniform negative charge, while the reducing agent ensures that the proteins remain denatured and linear.

SDS-PAGE

Gel Preparation:

Casting SDS-PAGE Gel: A polyacrylamide gel is prepared for SDS-PAGE, with the concentration of acrylamide varying depending on the size range of proteins to be analyzed. The gel typically consists of a stacking gel and a separating gel.

SDS-PAGE Run:

Overlaying the IEF Gel: The equilibrated IEF gel is placed on top of the SDS-PAGE gel. The interface between the two gels must be carefully aligned to ensure effective transfer of proteins.

Electrophoresis: An electric field is applied, causing proteins to migrate through the polyacrylamide gel. Smaller proteins travel faster and further through the gel matrix, while larger proteins move more slowly, resulting in separation based on size.

Staining and Visualization

Staining:

After electrophoresis, the gel is stained to visualize the separated proteins. Common staining methods include Coomassie Brilliant Blue, which binds to proteins and provides a blue coloration, and silver staining, which offers higher sensitivity for detecting low-abundance proteins.

Destaining:

Excess stain is removed from the gel to enhance the contrast of the protein bands. This is achieved by washing the gel in a destaining solution, which removes background staining and highlights the protein spots.

Image Analysis and Data Interpretation

Gel Imaging:

The stained gel is scanned or photographed using a gel imaging system. This produces a digital image of the gel, which is used for further analysis.

Image Analysis:

Advanced image analysis software is used to detect, quantify, and analyze the protein spots on the gel. The software compares the spots between different gels or conditions to identify changes in protein expression or modifications.

Data Interpretation:

Spot Identification: Protein spots are identified using databases and tools that match spot patterns with known protein sequences. This may involve mass spectrometry for detailed protein identification and characterization.

The principle of two-dimensional gel electrophoresis (Hiller-Sturmhöfel et al., 2008).

Advantages of 2D Gel Electrophoresis

• High Resolution: By separating proteins in two dimensions, 2DGE achieves superior resolution compared to one-dimensional techniques. This allows for the analysis of complex protein mixtures and the detection of subtle differences in protein expression and modifications.
• Comprehensive Protein Profiling: The technique enables the identification of a wide range of proteins from a single sample, providing a comprehensive overview of the proteome.
• Post-Translational Modification Analysis: 2DGE is particularly effective in studying post-translational modifications, such as phosphorylation and glycosylation, due to its sensitivity in detecting differences in pI.
• Quantitative Analysis: 2DGE can be coupled with image analysis software to quantify protein expression levels, facilitating comparative studies between different conditions or treatments.

Disadvantages of 2D Gel Electrophoresis

• Complexity and Variability: The technique is complex and requires meticulous handling of samples, which can lead to variability in results. Factors such as gel preparation, sample loading, and running conditions must be carefully controlled.
• Limited Detection of Low-Abundance Proteins: Proteins present in low abundance may be masked by more abundant proteins, making them difficult to detect and analyze.
• Resolution Limitations: While 2DGE provides high resolution, it may not fully resolve all proteins, especially those with similar pI or molecular weight.
• Time-Consuming: The process is labor-intensive and time-consuming, involving multiple steps and requiring significant expertise.

Issues in 2D Gel Electrophoresis and Their Solutions

Poor Resolution of Protein Spots

Problem:

Poor resolution can result from several factors, including incomplete focusing during IEF or inadequate separation during SDS-PAGE.

Solutions:

• Optimize IEF Conditions: Ensure the pH gradient covers the full range of pIs for the proteins of interest. Use high-quality ampholytes and maintain appropriate voltage and running times to achieve thorough focusing.
• Improve Sample Preparation: Ensure complete solubilization of proteins by using appropriate lysis buffers and detergents. Reduce and alkylate disulfide bonds to prevent protein aggregation.
• Optimize SDS-PAGE Conditions: Adjust the concentration of acrylamide in the gel to match the size range of the proteins being analyzed. Use a gradient gel if the sample contains a wide range of protein sizes.

Streaking and Smearing of Protein Spots

Problem:

Streaking and smearing can obscure protein spots and complicate data interpretation. This issue is often caused by sample overloading, salt contamination, or protein degradation.

Solutions:

• Avoid Sample Overloading: Use an appropriate amount of protein for the gel size. Overloading can lead to streaking and poor resolution.
• Desalt Samples: Remove excess salts using desalting columns or dialysis before applying samples to the gel.
• Use Protease Inhibitors: Add protease inhibitors to the lysis buffer to prevent protein degradation during sample preparation.

Horizontal or Vertical Streaking

Problem:

Horizontal streaking typically occurs during IEF, while vertical streaking occurs during SDS-PAGE. These streaks can interfere with protein spot identification.

Solutions:

• Optimize IEF Running Conditions: Ensure the IEF gel has a stable pH gradient. Use proper voltage steps to prevent horizontal streaking.
• Ensure Uniform Gel Casting: Cast the SDS-PAGE gel carefully to avoid uneven polymerization, which can cause vertical streaking.
• Improve Protein Solubilization: Use chaotropic agents and detergents to fully solubilize proteins and reduce horizontal streaking.

Incomplete Transfer Between Dimensions

Problem:

Incomplete transfer of proteins from the IEF gel to the SDS-PAGE gel can result in missing spots or faint bands.

Solutions:

• Ensure Proper Equilibration: Equilibrate the IEF gel thoroughly in SDS-containing buffer to ensure proteins are fully coated with SDS before SDS-PAGE.
• Align Gels Correctly: Carefully overlay the IEF gel on the SDS-PAGE gel to ensure proper contact and transfer.
• Optimize Electrophoresis Conditions: Adjust the running buffer and electrophoresis conditions to ensure efficient protein transfer.

Protein Precipitation

Problem:

Protein precipitation during sample preparation or IEF can lead to loss of protein and poor reproducibility.

Solutions:

• Optimize Sample Buffer: Use buffers that maintain protein solubility. Include urea, thiourea, and appropriate detergents to prevent precipitation.
• Maintain Proper Sample Handling: Avoid freeze-thaw cycles and prolonged storage of samples. Store samples at appropriate temperatures with stabilizing agents.

Gel Reproducibility Issues

Problem:

Reproducibility issues can arise from variations in gel casting, electrophoresis conditions, and sample preparation.

Solutions:

• Standardize Protocols: Develop and adhere to standardized protocols for gel preparation, electrophoresis, and staining.
• Use High-Quality Reagents: Ensure consistent quality of reagents and materials used in the experiment.
• Automate Steps Where Possible: Use automated systems for gel casting, sample application, and electrophoresis to minimize human error.

Difficulty in Spot Detection and Analysis

Problem:

Detecting and analyzing protein spots can be challenging due to variations in spot intensity and background noise.

Solutions:

• Use Sensitive Staining Methods: Employ highly sensitive staining techniques like silver staining or fluorescence-based methods to enhance spot visibility.
• Optimize Image Acquisition: Use high-resolution imaging systems and adjust exposure settings to capture clear images of protein spots.
• Utilize Advanced Software: Apply sophisticated image analysis software to accurately detect and quantify protein spots, reducing subjective interpretation.

Inconsistent pH Gradient

Problem:

An inconsistent pH gradient can result in poor focusing and resolution of proteins during IEF.

Solutions:

• Use High-Quality Ampholytes: Ensure the use of reliable and high-quality ampholytes to create a stable and reproducible pH gradient.
• Monitor Gel Aging: Be aware that gels can degrade over time, affecting the pH gradient. Use fresh gels whenever possible.

Difference Between One-Dimensional, Two-Dimensional Electrophoresis, and SDS-PAGE

Reference

1. Hiller-Sturmhöfel, Susanne, Josip Sobin, and R. Dayne Mayfield. "Proteomic approaches for studying alcoholism and alcohol-induced organ damage." Alcohol Research & Health 31.1 (2008): 36.