Lecture_6_notes_Protein_Techniques_BCH400_Spring_2025

Chapter 5 Techniques in Protein Biochemistry

Overview of the Chapter

This chapter focuses on various essential techniques in protein biochemistry, crucial for studying proteins' structure, function, and applications in research and medicine. Topics include:

• Monoclonal antibodies

• Western blotting

• Enzyme-Linked Immunosorbent Assays (ELISAs)

Questions for Review

For a comprehensive understanding, refer to questions on page 100 of the 4th edition, specifically questions: 2, 7, 12, 14, 15, 19, 21, and 22. Access the chapter through WebCampus.

Importance of Protein Purification

Why Purify Proteins?

The purification of proteins is fundamental in biochemistry and molecular biology due to various reasons:

• Studying Enzyme Functions: Pure proteins allow researchers to analyze enzyme kinetics, reaction mechanisms, and interactions with substrates and inhibitors, essential for drug development and biochemical pathways understanding.

• Structural Analysis: Techniques such as x-ray crystallography and NMR spectroscopy require high-purity proteins to derive accurate structural data that inform about molecular mechanisms and drug design.

• Determining Post-Translational Modifications: Understanding how proteins are modified after synthesis is crucial for functional studies, revealing regulatory mechanisms and interactions.

Key Quote

Efraim Racker, a pivotal figure in biochemistry, stated: "Don’t waste clean thinking on dirty enzymes." He began his career at the Austrian Academy of Art before achieving his MD in 1938, highlighting the value of purity in research.

General Steps in Protein Purification

The protein purification process generally follows these steps:

1. Develop an Assay: Create a method for detecting or quantifying the protein of interest.

2. Choose a Source of Protein: Select the biological source from which proteins will be extracted (e.g., tissues, cell cultures).

3. Prepare Tissue Extract: Employ methods including:

   • Cell Disruption: Breaking open cells to release proteins.

   • Subcellular Fractionation: Isolating different cellular components.

   • Protein Fractionation: Applying multiple techniques to further purify proteins.

4. Determine Purity: Assess the purity of the preparation through techniques such as SDS-PAGE or spectrophotometry.

Fractionation by Differential Centrifugation

This technique is vital for separating cellular components based on size and density:

• Centrifuge at 600 g: The pellet contains unbroken cells, while the supernatant contains soluble proteins.

• Centrifuge at 15,000 g: The pellet captures organelles, including nuclei and chloroplasts, while further purification occurs in the supernatant.

• Centrifuge at 100,000 g: Isolates mitochondria, with the remaining supernatant containing a microsomal fraction (rough and smooth endoplasmic reticulum, Golgi apparatus, lysosomes).

• The final supernatant becomes the cytosol, containing soluble enzymes.

Salting Out Technique

Salting out is a widely used strategy in protein purification that capitalizes on the solubility characteristics of proteins:

• Salting In: Most proteins require a certain concentration of salt to remain in solution.

• Salting Out: Increasing the salt concentration leads to precipitation of proteins based on their solubility. This is commonly performed with ammonium sulfate, which allows the separation of proteins through successive addition and centrifugation.

Dialysis for Salt Removal

After the salting out process, dialysis is often employed to remove excess salt:

• Proteins are placed in a cellophane bag that permits the passage of small salt ions but retains larger protein molecules. This is crucial to ensure that excess salt does not interfere with subsequent experimental setups.

Gel-Filtration/Size Exclusion Chromatography

This method relies on a porous gel that separates proteins based on size:

• Small Molecules: Enter the gel's pores and elute later.

• Large Molecules: Are excluded from the pores and elute first, allowing for effective separation based on size.

Ion-Exchange Chromatography

This technique exploits the charge properties of proteins:

• Anion Exchange Chromatography: Uses a positively charged resin to bind negatively charged proteins.

• Cation Exchange Chromatography: Utilizes a negatively charged resin for positively charged proteins.

• Proteins are eluted based on their charge, leading to effective separation.

Affinity Chromatography

This method is based on the specific interactions between proteins and ligands:

• Separation: Proteins bind to a resin that has a ligand specific for that protein, allowing for targeted purification.

• Elution: Proteins can be eluted by changing the ionic environment or by adding competing ligands.

Electrophoresis Techniques

Electrophoresis methods are crucial for analyzing protein samples:

• SDS-PAGE: Proteins are subject to polyacrylamide gel electrophoresis in the presence of SDS, which denatures proteins by imparting a negative charge, allowing separation based on size.

• Isoelectric Focusing: Separates proteins based on their isoelectric point (pI), where proteins migrate until they reach a point of neutral charge.

• 2-D Electrophoresis: Combines both isoelectric focusing and SDS-PAGE to separate proteins based on charge and size, providing a comprehensive protein profile.

Detection Methods Post-Electrophoresis

After electrophoresis, various techniques can be used to detect proteins, including:

• Protein Stains: Visualize the proteins in gel.

• Radioactive Labels: Provide sensitive detection methods.

• Western Blots: Specific protein detection using antibodies.

Advanced Techniques in Protein Analysis

• High-Performance Liquid Chromatography (HPLC): Used for precise separation and analysis of proteins.

• Edman Degradation: A method to determine amino acid sequences from the N-terminal end of proteins.

• Mass Spectrometry: Techniques such as MALDI-TOF measure mass-to-charge ratios for protein identification.

Activity Measurement and Purity Assessment

To assess protein purity and activity, one can evaluate a specific enzyme, such as Alcohol Dehydrogenase:

• Reaction: The conversion of ethanol to acetaldehyde and NAD+ to NADH can be monitored.

• Specific Activity Calculation: Defined as Specific Activity = Total Activity / Total Protein, which is essential for evaluating the effectiveness and purity of the enzyme preparation.

Methods to Determine Protein Structure

Techniques to elucidate protein structure include:

• X-ray Crystallography: Provides high resolution and detailed diffraction patterns for structural data.

• 2-D Nuclear Magnetic Resonance (NMR): Reveals information based on distances between atoms within the protein structure.

Immunoglobulin G (IgG) Structure and Function

IgG is a Y-shaped hetero-tetrameric protein, playing pivotal roles in immune responses:

• Mechanism: Antibody binding can disable pathogens or tag them for immune response elimination.

• Applications: Extensive uses in therapy including cancer treatment and inhibiting blood clotting.

Monoclonal Antibodies in Therapy

Monoclonal antibodies gained substantial market share in 2021 as effective therapeutic agents for various conditions, primarily cancers:

• scFv Fragments: Rapidly emerging due to their small size and multivalent structures, enhancing therapeutic potential.

Epitopes and Antibody Production

Epitopes

Epitopes, typically around 6 amino acids in size, are specific regions recognized by antibodies:

• B-cell Production: Each B-cell is programmed to produce antibodies that target a specific epitope, highlighting the specificity of the immune response.

Antibody Harvesting Techniques

Different methods are used to harvest antibodies:

• Polyclonal Antibodies: Obtained through methods like tail bleeding; these antibodies are a mix recognizing multiple epitopes.

• Monoclonal Antibodies: Acquired from tissue culture supernatant or ascitic fluid, they are identical and target a specific epitope.

Western Blotting Technique

Western blotting is crucial for identifying specific proteins in complex mixtures:

• Process: Proteins are separated via SDS-PAGE, transferred to nitrocellulose or PVDF membranes, and detected using specific antibodies.

Enzyme-Linked Immuno-Sorbent Assays (ELISAs)

ELISAs are vital for detecting antibodies in various samples, such as blood, and play a crucial role in HIV testing, often alongside Western blot testing.