CHM 821 Assignment

Two-Dimensional Electrophoresis
Two-Dimensional Electrophoresis powerful separation techniques used to achieve higher

resolution and better separation of complex mixtures of proteins. It combines two different
separation methods, often isoelectric focusing (IEF) and Sodium dodecyl Sulfate-polyacrylamide
gel Electrophoresis (SDS-PAGE), to enhance the separation of protein based on both their
isoelectronic points (pI) and molecular weights.
Working Principle of 2-DE

1. Isoelectric focusing is the first dimension of 2DE and involves separating proteins based on
their isoelectric point (pI), which is the pH at which a protein has no net charge. The setup
consists of a gel containing an immbolized pH gradient. Proteins loaded onto the gel strip, and an
electric field is applied across the gel. The proteins migrate towards their respective isoelectric
points, where they become focused and stop moving due to their net charge becoming zero. This
creates a separation based on charge.
2. Sodium Dodecyl Sulfate-Polyacrylamide Gel electrophoresis (SDS-PAGE): The second
dimension involves separating proteins based on their molecular weight. The gel strip containing
focused proteins from the IEF step is placed onto the top of an SDS-PAGE gel. SDS is a
detergent that denaturates proteins and imparts a uniform negative charge to them, which imparts
their original charge. An electric field is applied again, causing the proteins to migrate through
the gel based on their molecular weight. Smaller proteins move faster and farther, while larger
proteins move more slowly and remain closer to the starting point.
Figure 1.1 Schematic representation of the

2DE
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In the figure above, the proteins initially loaded onto the isoelectric focusing gel move towards
their isoelectric points. After focusing, the gel strip is placed on top of the SDS-PAGE gel, and a
second electrophoresis is conducted to separate the proteins based on their molecular weight. The
result is a 2D gel Image with protein spots each representing a unique protein in the mixture. The
horizontal axis represents isoelectric point, and the vertical axis represents molecular weight.
This technique is widely used in proteomics research to analyze protein expression patterns,
identify post-transitional modifications and compare proteins profiles across different samples.
Advantages of 2-DE
1. Advantages of 2-DE allows for the separation of thousands of protein from a complex
mixtures, providing higher resolution compared to one-dimensional Techniques like
SDS-PAGE.
2. Comprehensive analysis: it enables the identification of proteins that have different
isoforms or post-translational modifications, offering a more complete picture of protein
expression patterns.
3. Protein Profiling: by mapping the positions of proteins on the 2-DE gel, researchers can
create protein profiles for different samples, aiding in comparative analysis between
different conditions or biological samples.
4. Protein Complexes: Can help identify complexes and interactions by resolving proteins
that co-migrate in different dimensions. This is valuable for understanding cellular
processes.
5. Diseases Studies: It’s widely used in proteomics studies to investigate changes in protein
expression patterns associated with diseases, helping to identify potential biomarkers.
Disadvantages:
1. Labor-Intensive: technique is time- consuming and requires careful sample preparation,
gel casting, running, standing, and analysis, making it less amenable to high throughout..
2. Technical Challenges: achieving consistent and reproducible results can be challenging
due to factors such as variations in gel pH, temperature and gel handling.
3. Sample loss and variability: there can be protein loss during the different steps of the
process, leading to variability between replicates.
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4. Difficulty to Identifying Low Abundance proteins: Despite its high resolution, 2-DE
might still Struggle to detect low.
5. Focusing Gradient issues: isoelectric Focusing gradients might not cover the entire pH
range, leading to incomplete separation of proteins with extreme pI values.
Application of Two-Dimensional Electrophoresis
Two-Dimensional electrophoresis can be applied in the following areas which include:
Proteomics
2DE is widely used in proteomics research to analyze the protein content of a sample. It enables
the separation and identification of thousands of proteins in a complex mixture, providing
insights into protein expression levels, post-translational modifications, and interactions. This is
particularly valuable for studying disease mechanisms, biomarker discovery, and understanding
cellular responses.
Clinical Research and Diagnostics
In the medical field, 2DE can help identify disease-associated biomarkers, aiding in the diagnosis
and monitoring of various conditions. It has been applied to study diseases such as cancer,
neurological disorders, cardiovascular diseases, and infectious diseases, providing valuable
information about protein expression changes.
Pharmaceutical Research
Two-dimensional electrophoresis is employed in drug development and pharmacology to analyze
the effects of drugs on protein expression and post-translational modifications. This can help
researchers understand drug mechanisms of action, identify potential off-target effects, and
optimize drug candidates.
Plant and Microbial Science
2DE is used to analyze the proteomes of plants and microorganisms, providing insights into how
they respond to environmental changes, stressors, and genetic modifications. It helps researchers
understand plant-microbe interactions, crop improvement, and the development of microbial-
based biotechnologies.
Toxicology
Researchers use 2DE to investigate the effects of toxins, pollutants, and chemicals on protein
expression in cells and tissues. This information is valuable for assessing the potential toxicity of
substances and understanding their mechanisms of action.
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Food Science
Two-dimensional electrophoresis is applied in food science to analyze the protein content of
different foods, helping to ensure quality control, detect allergens, and assess the effects of
processing on protein composition.
Environment studies
Environmental scientists use 2DE to study protein expression changes in response to
environmental stressors, pollutants, and changes in habitat. This information contributes to a
better understanding of ecosystem health and responses to environmental changes.
Forensic Science
In forensic investigations, 2DE can be used to analyze biological samples (e.g., blood, tissue,
hair) and help identify individuals or determine factors such as post-mortem interval.
Comparative Studies
Researchers use 2DE to compare protein expression patterns between different samples, such as
healthy vs. diseased tissues or control vs. experimental conditions. This approach can provide
insights into molecular changes associated with various physiological states.
Overall, two-dimensional electrophoresis is a versatile technique that has applications across
diverse scientific disciplines, helping researchers gain a deeper understanding of complex
biological systems and their responses to various stimuli.
REFERENCES
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Gorg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe B., (2000). The current state of two-
dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 21(16),
1037-1053.
Klose J, (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse
tissues, Journal of biological Chemistry, 250(10), 4007-4021.
O’Farrell, P.H. (1975). Higher resolution two-dimensional electrophoresis of proteins. The
Journal of Biological Chemistry, 250(10), 4007-4021.
Rabilloud, T., & Lelong, C. (2011). Two dimensional gel electrophoresis in proteomics: a tutorial.
Journal of Proteomics, 74(10), 1829-1841
Righetti, P. G., & Castagna, A. (2003). Two-dimensional gel maps and databases of the human
macrophage cell line U937. Electrophoresis, 24(14), 2356-2365

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Two-Dimensional Electrophoresis

Two-Dimensional Electrophoresis powerful separation techniques used to achieve higher

Working Principle of 2-DE

Figure 1.1 Schematic representation of the

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