Course Overview:

This course will cover various techniques used for separating proteins based on their size, charge, and other properties.

Prerequisites:

Basic knowledge of biochemistry and molecular biology.

Introduction to Protein Separation Techniques

Proteomics is the study of proteins, including their structures, functions, and interactions. Protein separation techniques are essential in proteomics to isolate and identify proteins from complex biological samples. Here’s an overview of common protein separation techniques used in proteomics:

1. Gel Electrophoresis:
   • Principle: Gel electrophoresis separates proteins based on their size and charge.
   • Types:
     • SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis): Denatures proteins and separates them based on size.
     • 2D-PAGE (Two-Dimensional Gel Electrophoresis): Combines isoelectric focusing (separation based on charge) with SDS-PAGE to separate proteins based on both size and charge.
   • Applications: Protein separation, quantification, and visualization of protein patterns.
2. Liquid Chromatography (LC):
   • Principle: LC separates proteins based on their interactions with a stationary phase and a mobile phase.
   • Types:
     • Reverse-Phase Chromatography (RPC): Separates proteins based on hydrophobicity.
     • Ion Exchange Chromatography (IEX): Separates proteins based on charge.
     • Size Exclusion Chromatography (SEC): Separates proteins based on size.
   • Applications: Protein purification, quantification, and identification.
3. Mass Spectrometry (MS):
   • Principle: MS identifies proteins based on their mass-to-charge ratio (m/z).
   • Types:
     • MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight): Ionizes proteins using a laser and measures their m/z ratio.
     • ESI-MS (Electrospray Ionization Mass Spectrometry): Ionizes proteins in solution and measures their m/z ratio.
   • Applications: Protein identification, quantification, and characterization.
4. Immunoaffinity Chromatography:
   • Principle: Uses antibodies to selectively bind and isolate proteins of interest.
   • Applications: Purification of specific proteins or protein complexes.
5. Protein Microarrays:
   • Principle: Arrays of immobilized proteins are used to study protein-protein interactions, protein-DNA interactions, and protein-ligand interactions.
   • Applications: Protein interaction studies, biomarker discovery, and drug discovery.
6. Capillary Electrophoresis (CE):
   • Principle: CE separates proteins based on their charge and size in a capillary tube.
   • Applications: Protein analysis, including protein quantification and purity determination.

These protein separation techniques are essential tools in proteomics research, enabling the identification, quantification, and characterization of proteins in complex biological samples.

Principles of protein separation based on size, charge, and other properties

Protein separation techniques are based on various properties of proteins, including size, charge, hydrophobicity, and binding affinity. Here’s an overview of the principles of protein separation based on these properties:

1. Size-Based Separation:
   • Gel Electrophoresis: Separates proteins based on size using an electric field. Smaller proteins migrate faster through the gel, while larger proteins migrate more slowly.
   • Size Exclusion Chromatography: Separates proteins based on their size in solution. Larger proteins elute first, while smaller proteins are retained longer.
2. Charge-Based Separation:
   • Ion Exchange Chromatography: Separates proteins based on their net charge at a specific pH. Positively charged proteins (cation exchange) or negatively charged proteins (anion exchange) interact with charged groups on the stationary phase.
   • Isoelectric Focusing (IEF): Separates proteins based on their isoelectric point (pI), the pH at which the protein has no net charge. Proteins migrate to their pI and stop moving.
3. Hydrophobicity-Based Separation:
   • Hydrophobic Interaction Chromatography (HIC): Separates proteins based on their hydrophobicity. Proteins with higher hydrophobicity interact more strongly with the hydrophobic stationary phase and elute later.
4. Binding Affinity-Based Separation:
   • Affinity Chromatography: Separates proteins based on specific interactions between a ligand immobilized on the stationary phase and the protein of interest. Proteins with high affinity for the ligand are retained, while others are washed away.
5. Other Properties:
   • Gel Filtration Chromatography (GFC): Also known as size exclusion chromatography, separates proteins based on their size but can also separate based on shape and hydration.
   • Electrophoretic Mobility Shift Assay (EMSA): Separates proteins based on their ability to bind to DNA, RNA, or other ligands.

These principles form the basis of protein separation techniques used in various fields, including biochemistry, molecular biology, and proteomics, and are essential for studying protein structure, function, and interactions.

Factors influencing protein separation efficiency

Several factors can influence the efficiency of protein separation techniques. These factors should be considered and optimized to achieve the best results:

1. Matrix or Support Material: The choice of matrix or support material in chromatography columns (e.g., agarose, sepharose, silica) can affect the binding capacity, resolution, and efficiency of protein separation.
2. Column Size and Geometry: The size and geometry of the chromatography column can influence the flow rate, resolution, and capacity of the column, impacting the efficiency of protein separation.
3. Buffer Composition and pH: The buffer composition and pH can affect the stability and solubility of proteins, as well as the interactions between proteins and the stationary phase in chromatography.
4. Temperature: The temperature can affect the stability and conformation of proteins, as well as the kinetics of protein-protein interactions, potentially impacting the efficiency of protein separation.
5. Flow Rate: The flow rate of the mobile phase in chromatography can influence the contact time between proteins and the stationary phase, affecting the efficiency of protein separation.
6. Sample Preparation: The method used for sample preparation (e.g., cell lysis, protein extraction) can affect the quality and quantity of proteins in the sample, impacting the efficiency of protein separation.
7. Protein Concentration: The concentration of proteins in the sample can affect the binding capacity and resolution of the separation technique, influencing the efficiency of protein separation.
8. Presence of Contaminants: The presence of contaminants in the sample (e.g., nucleic acids, lipids) can interfere with protein separation, reducing the efficiency of the technique.
9. Choice of Separation Technique: The choice of protein separation technique (e.g., gel electrophoresis, chromatography) and the specific method within that technique can impact the efficiency of protein separation.
10. Instrumentation and Equipment: The quality and condition of the instrumentation and equipment used for protein separation can affect the reproducibility and efficiency of the separation technique.

By optimizing these factors, researchers can improve the efficiency and effectiveness of protein separation techniques, leading to better results and insights into protein structure and function.

Gel Electrophoresis

Principles of gel electrophoresis: SDS-PAGE, native PAGE

Gel electrophoresis is a widely used technique in biochemistry and molecular biology to separate and analyze proteins based on their size and charge. There are two main types of gel electrophoresis: SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) and native PAGE (Polyacrylamide Gel Electrophoresis).

SDS-PAGE:

1. Principle: SDS-PAGE separates proteins based on their molecular weight. SDS (Sodium Dodecyl Sulfate) denatures proteins and binds to them, imparting a negative charge proportional to the protein’s mass. Therefore, the migration rate of the protein through the gel is primarily determined by its size.
2. Procedure:
   • Proteins are first denatured by heating in the presence of SDS, which unfolds the proteins and coats them with negatively charged SDS molecules.
   • The SDS-treated proteins are then loaded into the wells of a polyacrylamide gel along with a molecular weight marker.
   • An electric current is applied to the gel, causing the proteins to migrate through the gel towards the positive electrode. Smaller proteins move faster and migrate farther than larger proteins.
   • After electrophoresis, the gel is stained with a dye (e.g., Coomassie Blue) to visualize the separated proteins.

Native PAGE:

1. Principle: Native PAGE separates proteins based on their size and charge without denaturing them. The migration of proteins is influenced by their size, shape, charge, and interactions with the gel matrix.
2. Procedure:
   • Proteins are mixed with a non-denaturing buffer that maintains their native conformation and charge.
   • The protein mixture is loaded into the wells of a polyacrylamide gel without SDS.
   • An electric current is applied to the gel, causing the proteins to migrate based on their size and charge.
   • After electrophoresis, the gel is typically stained with a dye or used for further analysis, such as protein-protein interaction studies.

Applications:

• SDS-PAGE is commonly used for protein purification, quantification, and analysis of protein complexes.
• Native PAGE is useful for studying native protein structure, protein-protein interactions, and protein activity.

Considerations:

• Both techniques require careful sample preparation, gel casting, and running conditions to achieve optimal separation.
• Gel percentage and running conditions can be adjusted to improve resolution for specific protein sizes or applications.

Sample preparation for gel electrophoresis

Sample preparation for gel electrophoresis depends on the type of gel electrophoresis being performed (e.g., SDS-PAGE, native PAGE) and the nature of the samples (e.g., purified proteins, cell lysates). Here’s a general overview of sample preparation for gel electrophoresis:

1. Protein Samples:
   • Purified Proteins: If working with purified proteins, mix the protein sample with an appropriate sample buffer (e.g., Laemmli buffer for SDS-PAGE) containing SDS and a reducing agent (e.g., DTT, β-mercaptoethanol) to denature the proteins and break disulfide bonds.
   • Cell Lysates: If working with cell lysates, first prepare the lysate using a suitable lysis buffer to release proteins from cells. Then, mix the lysate with an appropriate sample buffer for denaturation and loading onto the gel.
2. Denaturation:
   • Heat the protein samples at 95-100°C for 5-10 minutes to denature the proteins and ensure uniform binding of SDS.
3. Loading:
   • Load the protein samples into the wells of the gel using a micropipette. Include a molecular weight marker for reference.
4. Running the Gel:
   • Place the gel cassette into the electrophoresis tank filled with running buffer (e.g., Tris-glycine buffer for SDS-PAGE).
   • Apply an electric current according to the manufacturer’s instructions and run the gel until the tracking dye reaches the bottom of the gel.
5. Visualization:
   • After electrophoresis, visualize the separated proteins using a staining method appropriate for the gel type (e.g., Coomassie Blue staining for SDS-PAGE).
6. Analysis:
   • Analyze the gel using imaging equipment (e.g., gel documentation system) to capture images of the separated proteins.
   • Use appropriate software for quantification and analysis of the protein bands.

Notes:

• For native PAGE, omit the denaturation step and use a non-denaturing sample buffer.
• Care should be taken to prevent protein degradation and contamination during sample preparation.
• Samples should be handled carefully to avoid introducing bubbles or other artifacts that may affect the gel run.

Hands-on demonstration of SDS-PAGE

To conduct a hands-on demonstration of SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), you can use a protein sample such as a commercial protein ladder or prepared sample mixed with SDS and a reducing agent. Here’s a basic outline for the demonstration:

Materials Needed:

• Mini gel electrophoresis system with casting stand and combs
• Acrylamide/bis-acrylamide solution, ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) for gel preparation
• Tris-glycine running buffer
• Protein sample (e.g., BSA, commercial protein ladder)
• SDS-PAGE sample buffer (Laemmli buffer) with reducing agent (e.g., β-mercaptoethanol)
• Heat block or water bath for protein denaturation
• Power supply for electrophoresis
• Coomassie Blue staining solution
• Destaining solution

Procedure:

1. Prepare the Gel:
   • Prepare a separating gel (e.g., 12% acrylamide) and a stacking gel (e.g., 4% acrylamide) using the appropriate acrylamide/bis-acrylamide solution, APS, TEMED, and running buffer.
   • Assemble the gel sandwich with the casting stand and combs, and allow the gel to polymerize.
2. Prepare the Protein Sample:
   • Mix the protein sample with SDS-PAGE sample buffer and a reducing agent.
   • Heat the sample at 95-100°C for 5-10 minutes in a heat block or water bath to denature the proteins.
3. Load the Gel:
   • Carefully remove the combs and rinse the wells with running buffer.
   • Load the protein sample and a protein ladder into the wells using a micropipette.
4. Run the Gel:
   • Place the gel in the electrophoresis tank and fill it with running buffer.
   • Connect the electrodes to the power supply and run the gel at a constant voltage (e.g., 100-150V) until the tracking dye reaches the bottom of the gel.
5. Stain and Visualize the Gel:
   • Remove the gel from the electrophoresis tank and carefully disassemble the gel sandwich.
   • Stain the gel with Coomassie Blue staining solution for about 1 hour.
   • Destain the gel with destaining solution until protein bands are visible.
6. Analysis:
   • Visualize the protein bands and compare them to the protein ladder to estimate the molecular weight of the proteins in the sample.

Safety Considerations:

• Wear appropriate personal protective equipment (lab coat, gloves, goggles).
• Handle acrylamide solutions with care, as they are toxic.
• Follow all laboratory safety guidelines and procedures.

This hands-on demonstration provides a practical and visual way for students to understand the principles and process of SDS-PAGE

Chromatographic Techniques (Part 1)

Principles of chromatography: ion exchange, size exclusion, affinity chromatography

Chromatography is a technique used to separate and analyze complex mixtures based on the differential interaction of components with a mobile phase and a stationary phase. Here’s an overview of the principles of three common types of chromatography:

1. Ion Exchange Chromatography:
   • Principle: Separates molecules based on their charge. The stationary phase contains charged groups that attract and bind molecules of opposite charge. The mobile phase (buffer) is used to elute the bound molecules by changing the pH or ionic strength.
   • Process:
     • Positively charged molecules (cations) bind to a negatively charged stationary phase (anion exchange) or vice versa.
     • The strength of binding depends on the charge of the molecule and the pH/ionic strength of the buffer.
   • Applications: Purification of proteins, nucleic acids, and other charged molecules.
2. Size Exclusion Chromatography (SEC):
   • Principle: Separates molecules based on their size and shape. The stationary phase contains porous beads that allow smaller molecules to enter the beads and elute later, while larger molecules pass through the column more quickly.
   • Process:
     • Larger molecules are excluded from the beads and elute first, while smaller molecules enter the beads and elute later.
     • The mobile phase (buffer) flows through the column, carrying the molecules through the stationary phase.
   • Applications: Separation of proteins, nucleic acids, and polysaccharides based on size.
3. Affinity Chromatography:
   • Principle: Separates molecules based on specific interactions between a ligand on the stationary phase and a target molecule. The ligand binds selectively to the target molecule, allowing other molecules to pass through the column.
   • Process:
     • The target molecule selectively binds to the ligand on the stationary phase.
     • After other molecules are washed away, the target molecule is eluted using a competitive ligand or by changing the buffer conditions.
   • Applications: Purification of proteins, antibodies, and other biomolecules based on specific interactions.

Overall, chromatography is a versatile technique used in various fields such as biochemistry, molecular biology, pharmaceuticals, and environmental science for the separation, purification, and analysis of complex mixtures. Each type of chromatography offers unique advantages and is selected based on the specific requirements of the experiment or application.

Protein separation using chromatography columns

Protein separation using chromatography columns involves the use of different chromatographic techniques to purify and separate proteins based on their specific properties. Here’s an overview of the process:

1. Column Preparation:
   • Choose a chromatography column appropriate for the desired separation technique (e.g., ion exchange, size exclusion, affinity).
   • Pack the column with the appropriate stationary phase (e.g., ion exchange resin, size exclusion beads, affinity ligand) according to the manufacturer’s instructions.
2. Sample Application:
   • Prepare the protein sample by solubilizing it in an appropriate buffer compatible with the chromatography technique.
   • Apply the protein sample to the column either by gravity flow or using a pump, ensuring the sample is evenly distributed across the column.
3. Washing:
   • Wash the column with a buffer to remove unbound proteins and contaminants, optimizing the washing conditions based on the specific chromatography technique and target protein.
4. Elution:
   • Elute the bound proteins from the column using an elution buffer that disrupts the interactions between the proteins and the stationary phase.
   • Collect the eluted fractions containing the purified proteins.
5. Analysis:
   • Analyze the eluted fractions using appropriate methods (e.g., SDS-PAGE, Western blotting, spectroscopy) to assess the purity and yield of the purified proteins.
6. Regeneration:
   • Regenerate the column by washing with cleaning solutions to remove any remaining bound proteins and regenerate the stationary phase for future use.
7. Storage:
   • Store the column according to the manufacturer’s instructions to maintain its integrity and performance for future use.

Overall, protein separation using chromatography columns is a versatile and widely used technique in biochemistry, molecular biology, and biotechnology for the purification and analysis of proteins with high purity and yield.

Hands-on session on column chromatography

A hands-on session on column chromatography can be a valuable learning experience for students to understand the principles and techniques involved in protein separation. Here’s a basic outline for conducting a hands-on session on column chromatography using an ion exchange resin:

Materials Needed:

• Chromatography columns (e.g., glass or plastic columns with stopcocks)
• Ion exchange resin (e.g., DEAE-Sepharose for anion exchange or CM-Sepharose for cation exchange)
• Buffers for equilibration, washing, and elution (e.g., Tris buffer, NaCl gradient)
• Protein sample (e.g., BSA or other protein standards)
• Graduated cylinders or pipettes for buffer and sample handling
• Collection tubes or fractions collector
• UV-visible spectrophotometer or protein assay kit for protein quantification
• Gloves, lab coats, and safety goggles

Procedure:

1. Column Preparation:
   • Pack the column with the ion exchange resin, ensuring it is properly packed and settled to avoid channeling.
   • Equilibrate the column with equilibration buffer to prepare it for sample application.
2. Sample Application:
   • Prepare the protein sample in the equilibration buffer.
   • Apply the sample to the column and collect the flow-through (unbound proteins).
3. Washing:
   • Wash the column with wash buffer to remove any remaining unbound proteins and contaminants.
4. Elution:
   • Elute the bound proteins from the column using an elution buffer containing a gradient of NaCl to disrupt the interactions between the proteins and the resin.
   • Collect fractions from the column and monitor protein elution using a UV-visible spectrophotometer or protein assay kit.
5. Analysis:
   • Analyze the fractions using a spectrophotometer or protein assay kit to determine protein concentration and purity.
   • Optionally, analyze the fractions using SDS-PAGE to visualize protein separation.
6. Discussion Points:
   • Principles of ion exchange chromatography and protein separation.
   • Factors influencing protein binding and elution.
   • Applications and limitations of column chromatography in protein purification.

Safety Considerations:

• Follow appropriate laboratory safety guidelines, including the use of gloves, lab coats, and safety goggles.
• Handle buffers and chemicals with care to avoid contamination and spills.

This hands-on session provides students with a practical understanding of column chromatography principles and techniques, enhancing their skills in protein separation and purification.

Chromatographic Techniques (Part 2)

High-performance liquid chromatography (HPLC) for protein separation

High-performance liquid chromatography (HPLC) is a powerful technique used for the separation, identification, and quantification of proteins and other biomolecules in complex mixtures. Here’s an overview of how HPLC can be used for protein separation:

1. Principle:
   • HPLC separates proteins based on their interactions with a stationary phase (packing material) and a mobile phase (solvent).
   • Proteins are eluted from the column based on their size, charge, hydrophobicity, or specific binding interactions with the stationary phase.
2. Column Selection:
   • HPLC columns for protein separation are typically packed with porous beads (e.g., silica or polymer) with a specific surface chemistry (e.g., C18 for hydrophobic interactions, ion exchange resins for charge-based separations).
3. Mobile Phase:
   • The mobile phase for protein separation is usually a buffered aqueous solution containing additives such as salts or organic solvents to optimize protein interactions with the stationary phase.
4. Sample Preparation:
   • Protein samples are typically denatured and reduced to break disulfide bonds and ensure uniform interactions with the stationary phase.
   • Samples may also be filtered or centrifuged to remove particulate matter that could clog the column.
5. Separation Modes:
   • Size Exclusion Chromatography (SEC): Separates proteins based on their size, with larger proteins eluting first.
   • Reverse Phase Chromatography (RPC): Separates proteins based on their hydrophobicity, with more hydrophobic proteins eluting later.
   • Ion Exchange Chromatography (IEX): Separates proteins based on their charge, with proteins of opposite charge to the stationary phase eluting first.
6. Detection:
   • Proteins can be detected using UV-visible absorption spectroscopy at 280 nm, which is absorbed by the aromatic amino acids in proteins.
   • Fluorescence detection can also be used for proteins that fluoresce naturally or are derivatized with a fluorescent label.
7. Applications:
   • HPLC is used in protein purification, characterization, and quality control in research, pharmaceuticals, and biotechnology.
   • It is also used for the analysis of post-translational modifications, protein-protein interactions, and protein folding studies.

Overall, HPLC is a versatile and powerful technique for protein separation, offering high resolution, sensitivity, and reproducibility, making it an essential tool in biochemistry and biotechnology.

Two-dimensional gel electrophoresis (2D-PAGE) for complex protein mixtures

Two-dimensional gel electrophoresis (2D-PAGE) is a powerful technique used to separate and analyze complex protein mixtures based on two different properties: isoelectric point (pI) and molecular weight. Here’s an overview of how 2D-PAGE works:

1. First Dimension – Isoelectric Focusing (IEF):
   • Proteins are separated based on their pI, which is the pH at which a protein carries no net charge.
   • A protein sample is loaded onto a gel strip with a pH gradient (e.g., pH 3-10) and subjected to an electric field.
   • Proteins migrate in the gel strip until they reach the pH equivalent to their pI, where they become focused into sharp bands.
2. Second Dimension – SDS-PAGE:
   • The gel strip from the first dimension is then placed on top of an SDS-PAGE gel.
   • Proteins are separated in the second dimension based on their molecular weight using SDS-PAGE, as described earlier.
   • The proteins migrate perpendicularly to the direction of the first dimension, resulting in a two-dimensional separation of proteins.
3. Visualization and Analysis:
   • After electrophoresis, the gel is typically stained with a protein stain (e.g., Coomassie Blue) to visualize the separated proteins.
   • The gel image is then analyzed using imaging software to detect and quantify protein spots.
   • Proteins of interest can be excised from the gel for further analysis, such as protein identification by mass spectrometry.

Advantages of 2D-PAGE:

• High resolution: Proteins are separated based on two independent properties, leading to better resolution compared to one-dimensional methods.
• Identification of protein isoforms: 2D-PAGE can separate protein isoforms with the same molecular weight but different pI values.
• Comparative analysis: It allows for the comparison of protein expression levels between different samples (e.g., diseased vs. normal tissue).

Applications of 2D-PAGE:

• Proteomics: Identification and quantification of proteins in complex mixtures.
• Biomarker discovery: Identification of proteins that are differentially expressed in disease states.
• Post-translational modification analysis: Detection of modified forms of proteins.

Overall, 2D-PAGE is a versatile technique for separating and analyzing complex protein mixtures, providing valuable insights into protein expression, modification, and function.

Proteomics isolation and separation methods. (A) Proteomics aims to compare the protein profiles across different samples, cell culture or whole tissue extracts. (B) Protein separation using gel electrophoresis. Spots from a 2D gel, or bands from a 1D gel, are excised for proteolysis. (C) Greater peptide yields are achieved by in-solution proteolysis. (D) Fractionation and enrichment strategies used for common peptides aim to reduce peptide complexity for mass spectrometric analysis.

Hands-on demonstration of HPLC and 2D-PAGE

Conducting hands-on demonstrations of HPLC and 2D-PAGE can be a challenging task due to the complexity and time-consuming nature of these techniques. However, here’s a simplified outline for each technique that can be adapted for educational purposes:

HPLC Demonstration:

Materials Needed:

• HPLC system (can be a simplified version or a demonstration kit)
• C18 column (or other suitable stationary phase)
• Protein samples (e.g., BSA, myoglobin)
• Mobile phase (e.g., buffer solution)
• UV-visible detector or other suitable detection method
• Solvents for column equilibration and washing
• Graduated cylinders or pipettes for buffer preparation
• Collection tubes for eluted fractions

Procedure:

1. Column Preparation:
   • Set up the HPLC system according to the manufacturer’s instructions.
   • Equilibrate the C18 column with the mobile phase.
2. Sample Preparation:
   • Prepare the protein samples in the mobile phase buffer.
3. Sample Injection:
   • Inject the protein sample into the HPLC system using an injection valve or syringe.
4. Separation:
   • Run the HPLC system at the appropriate flow rate and detection wavelength.
   • Monitor the elution of proteins from the column.
5. Data Analysis:
   • Collect and analyze the eluted fractions using the UV-visible detector.
   • Calculate the retention time and peak area of the proteins.

2D-PAGE Demonstration:

Materials Needed:

• Pre-cast 2D-PAGE gel (e.g., immobilized pH gradient gel)
• Protein samples (e.g., BSA, myoglobin)
• Isoelectric focusing (IEF) system
• SDS-PAGE system
• Staining solution (e.g., Coomassie Blue)
• Destaining solution
• Gel imaging system

Procedure:

1. Isoelectric Focusing (IEF):
   • Load the protein sample onto the IEF gel.
   • Perform isoelectric focusing to separate proteins based on their isoelectric point (pI).
2. Equilibration:
   • Equilibrate the IEF gel in SDS buffer to prepare it for SDS-PAGE.
3. Second Dimension (SDS-PAGE):
   • Transfer the IEF gel onto an SDS-PAGE gel.
   • Perform SDS-PAGE to separate proteins based on their molecular weight.
4. Staining and Visualization:
   • Stain the gel with Coomassie Blue to visualize the separated proteins.
   • Destain the gel and image it using a gel imaging system.

Discussion Points:

• Principles of HPLC and 2D-PAGE.
• Applications and significance of these techniques in protein separation and analysis.
• Comparison of the two techniques in terms of resolution, sensitivity, and complexity.

Note: These are simplified versions of the techniques and may not provide the same level of detail and resolution as actual experiments. Adjustments can be made based on the available equipment and resources.