Seminario 2A: técnicas utilizadas para el estudio de proteínas- Gabriel Scicolone

Introduction to Molecular Biology and Genetics Techniques

Overview of the Seminar

• Gabriel Cicolone introduces himself as a professor of molecular biology and genetics at UVA, alongside Sofía Martín. They will discuss techniques for analyzing proteins and nucleic acids during the seminar.

• The first part focuses on studies that allow detection, characterization, quantification, localization, and functional analysis of proteins. The second part will cover techniques for studying nucleic acids.

Sample Collection Methods

• Emphasizes the importance of sample collection in both experimental science and clinical settings; samples can be obtained from autopsies, biopsies, blood elements, or cultured cells.

• Discusses methods for preserving structure in samples (e.g., immunocytochemistry) versus those that do not require structural preservation (e.g., plasma or homogenized tissue).

Protein Analysis Techniques

Individual Protein Studies

• Highlights various techniques for studying individual proteins such as Western blotting, X-ray crystallography, and nuclear magnetic resonance spectroscopy.

Total Protein Analysis

• Introduces proteomics as a method to analyze all proteins within a biological system using mass spectrometry as a central element. Emphasis is placed on specific protein analysis techniques like immunohistochemistry and flow cytometry.

Gene Expression Relationship

Techniques Related to Gene Expression

• Discusses how certain techniques relate to gene expression by analyzing DNA, RNA, and proteins; specifically focusing on immunohistochemistry and fluorescence methods today. Flow cytometry is mentioned due to its use of antigen-antibody reactions similar to other discussed methods.

Immune Response Fundamentals

Antigen-Antibody Interaction

• Defines antigens as macromolecules or microorganisms that trigger an immune response when introduced into the body; examples include bacteria or specific proteins recognized by immune cells.

Immune Cell Functionality

• Describes how antigen-presenting cells (like macrophages) present antigens to lymphocytes which have antibodies on their surface that recognize these antigens specifically. This interaction leads to B cell activation and proliferation into memory B cells and plasma cells that produce antibodies against the antigen.

Antibody Structure

Composition of Antibodies

Understanding Antibodies and Lymphocytes

The Role of B Lymphocytes in Immune Response

• The plasma membrane of lymphocytes contains antibodies that are secreted by plasma cells; the variable portions of these antibodies differ among types, allowing them to recognize specific antigens.

• Each B lymphocyte has a unique antibody on its surface with a specific variable portion that recognizes only one antigenic determinant, highlighting the specificity of immune responses.

• Antigens, often macromolecules like proteins, initiate immune responses when introduced into an organism. Different regions within these proteins can be recognized by various antibodies.

• Distinct portions of an antigen recognized by antibodies are called epitopes or antigenic determinants; each epitope is specifically targeted by a corresponding antibody's variable region.

• An antigen can be recognized by multiple types of B lymphocytes, each having different antibodies with unique variable portions on their surfaces.

Clonal Expansion and Antibody Production

• In laboratory settings, animals such as rabbits are injected with antigens containing multiple epitopes to stimulate various B lymphocyte clones for antibody production.

• Upon introduction of an antigen into the body, it stimulates different B lymphocytes which proliferate and differentiate into memory cells and plasma cells.

• Each clone originates from a single B lymphocyte that recognizes a specific epitope; thus, many clones arise from exposure to an antigen with multiple determinants.

• This clonal expansion results in numerous plasma cells producing soluble antibodies targeting different epitopes from the same antigen, leading to polyclonal antibody production.

• The resulting serum contains polyclonal antibodies derived from various clones of plasma cells recognizing distinct antigenic determinants.

Monoclonal vs. Polyclonal Antibodies

• To obtain polyclonal antibodies in the lab, blood is collected and processed to separate plasma containing these antibodies for use in detecting target proteins.

• Monoclonal antibodies are designed to target a single epitope; they are produced by injecting an animal (commonly mice) with antigens containing multiple determinants.

• After immunization, various clones of B lymphocytes produce different antibodies; these are isolated and fused with tumor cells to create hybridomas capable of continuous growth.

• Hybridomas consist of fused normal B lymphocytes and immortal tumor cells; this fusion allows for sustained proliferation while maintaining antibody synthesis capabilities.

Antibody Types and Immunohistochemistry Techniques

Understanding Antibodies

• Polyclonal antibodies consist of various variable portions that recognize different antigenic determinants (epitopes) of the same antigen, while monoclonal antibodies are of a single type with one variable portion recognizing only one determinant.

Immunohistochemistry Technique Overview

• The immunohistochemistry technique allows for the localization of specific proteins within tissue samples, providing insights into their distribution and presence.

Protein Localization Process

• The process involves detecting a specific protein, such as a membrane receptor (e.g., epidermal growth factor receptor), to understand its cellular distribution in the studied tissue.

• Detection is achieved through an antigen-antibody reaction where antibodies recognize epitopes on the target protein. Polyclonal antibodies can bind to multiple epitopes, whereas monoclonal antibodies bind to a single epitope.

Sample Preparation for Immunohistochemistry

• To perform immunohistochemistry, it is crucial to preserve tissue structure by fixing samples quickly using substances that induce cell death while maintaining structural integrity.

• After fixation, tissues must be hardened for slicing into thin sections suitable for microscopic examination.

Visualization Techniques in Immunohistochemistry

• Once prepared, antibody solutions are applied to tissue sections; these antibodies will specifically bind to target proteins (e.g., epidermal growth factor receptor).

• To visualize antibody binding under a microscope, antibodies must be marked with detectable substances. This can include colorimetric markers or fluorochromes that emit visible light when excited.

Fluorescence Microscopy Applications

• Fluorochromes can be excited by ultraviolet light and emit radiation visible under fluorescence microscopy. This allows researchers to see where antibodies have bound in the tissue sample.

• Using fluorescence microscopy enables visualization of specific areas where the antibody-antigen complex exists due to emitted colors from excited fluorochromes.

Indirect Immunoassay Methodology

• An indirect method involves using an unmarked primary antibody that binds to the target antigen; this antibody is then recognized by a secondary labeled antibody produced in another animal (e.g., rabbit recognizing mouse monoclonal).

Immunohistochemistry and Immunofluorescence Techniques

Overview of Antibody Techniques

• The process involves producing secondary antibodies that recognize the constant portion of primary antibodies, allowing for the detection of specific proteins in tissues.

• Secondary antibodies are marked to identify those produced in a different species (e.g., rabbit recognizing mouse antibodies), enhancing specificity in immunoassays.

• Two methodologies are discussed: direct immunohistochemistry (where the primary antibody is marked) and indirect immunohistochemistry (where the secondary antibody is marked).

Methodological Applications

• Immunocytochemistry can be performed on cultured cells without cutting them, allowing for direct observation under a microscope using either marked primary or secondary antibodies.

• This technique enables the identification of proteins or glycoproteins within tissues or cells through antigen-antibody reactions, ensuring specificity for targeted proteins.

Distinctions Between Techniques

• Strictly speaking, "immunohistochemistry" refers to detecting antigens within tissue contexts, while "immunocytochemistry" focuses on cellular localization.

• The term "immunofluorescence" describes a type of immunoassay where fluorochromes are used as markers, requiring fluorescence microscopy for visualization.

Detection Methods

• Immunohistochemical techniques allow for antigen location detection via color changes from substances attached to antibodies; this can also involve fluorescent markers.

• Both methods can be direct (marked primary antibody) or indirect (marked secondary antibody), with examples provided illustrating various applications in cell cultures.

Examples and Observations

• In cell culture experiments, fluorescence indicates protein localization at the plasma membrane when using a green fluorochrome-marked secondary antibody.

• Specific proteins located in mitochondria and nuclei were identified using targeted antibodies, showcasing diverse cellular distributions observed through fluorescence microscopy.

• The technique's effectiveness relies on washing steps post-antibody binding to ensure only specifically bound antibodies remain visible during imaging.

Advanced Imaging Techniques

Immunofluorescence and Immunohistochemistry Techniques

Overview of Antibody Usage

• Two distinct antibodies are utilized: a contractin antibody made in mice and a Cox 4 enzyme antibody made in rabbits. This allows for the use of two different secondary antibodies, one targeting mouse antibodies (red fluorochrome) and another for rabbit antibodies (green fluorochrome).

Visualization Techniques

• The imaging reveals actin filaments in red, the Cox 4 enzyme around the nucleus in green, and DNA detected with a blue fluorochrome. Confocal microscopy is employed to focus on a single plane, eliminating images above and below.

Direct Immunofluorescence

• A direct immunofluorescence method is described where the primary antibody is directly labeled with a fluorochrome. This technique allows visualization of specific proteins within cells.

Example of Dual Staining

• An example shows DNA marked with DAPI (blue), proliferating cells marked by protein 67 (red), and tubulin (green). This illustrates another instance of immunofluorescence using confocal microscopy.

Indirect Immunofluorescence Methodology

• In indirect immunofluorescence, a secondary antibody recognizes the primary antibody that is linked to an enzyme (horseradish peroxidase). The reaction product from this enzyme creates a brown precipitate indicating where the antigen-antibody reaction occurred.

Comparing Immunofluorescence and Immunohistochemistry

Results Comparison

• Both techniques yield similar results regarding protein distribution; however, immunofluorescence focuses on individual cells while immunohistochemistry provides tissue-level insights.

Specificity of Techniques

• These methods are specific to particular proteins based on antigen-antibody reactions, allowing researchers to determine molecular locations within tissues or cells.

Western Blotting Technique

Introduction to Western Blotting

• Western blotting or immunoblotting is introduced as another methodology for identifying proteins based on antigen-antibody reactions but does not provide information about protein localization within tissues.

Sample Preparation Process

• The process begins with obtaining samples from homogenized tissues or plasma. This step ensures that proteins can be analyzed effectively through subsequent techniques like western blotting.

Gel Electrophoresis Explanation

• The first step involves gel electrophoresis which separates proteins by size. A gel acts as a filter allowing smaller proteins to migrate faster than larger ones under an electric current applied during the process.

Mechanism of Protein Separation

• Proteins are placed in an aqueous medium subjected to electrical charge; negatively charged proteins move towards the positive electrode while their migration speed depends on both size and charge characteristics.

Electrophoresis and Protein Separation Techniques

Introduction to Protein Separation

• The process begins with separating proteins in a gel based on their size, using a detergent called SDS that imparts negative charges to the proteins.

• SDS surrounds the proteins, allowing them to lose their three-dimensional structure and become linearized, which is essential for effective separation.

Preparing the Sample

• Proteins are extracted from tissue samples (e.g., plasma), often through homogenization, before being placed in a running buffer solution that facilitates charge and structural changes.

• The running buffer ensures proteins maintain a negative charge while losing tertiary and quaternary structures, preparing them for gel electrophoresis.

Gel Electrophoresis Process

• A polyacrylamide gel is used; its pore size varies with concentration, affecting protein migration rates during electrophoresis.

• Known molecular weight standards are loaded alongside unknown samples to allow for identification based on migration distance after applying an electric current.

Migration of Proteins

• Under electric field influence, larger proteins migrate slower than smaller ones; all move towards the positive pole due to their negative charges from SDS treatment.

• This method effectively separates proteins by molecular weight, visualizing distinct bands corresponding to different sizes post-electrophoresis.

Identifying Specific Proteins

• After separation, specific protein bands can be identified using antibodies that bind only to target proteins within the sample.

• An example includes identifying epidermal growth factor receptor (EGFR); primary antibodies bind to the target protein followed by secondary antibodies linked with enzymes for detection.

Transfer Techniques: Blotting

• To preserve antibody reactions without dispersion in gels, transferred solid surfaces like nitrocellulose membranes are utilized for better localization of reaction products.

• The transfer process involves applying an electric field again to move negatively charged proteins onto the membrane—a technique known as blotting.

Conclusion of Methodology

Western Blot Technique Overview

Introduction to Western Blotting

• The Western blot technique involves transferring proteins from a gel to a nitrocellulose membrane using an electric current, allowing for the analysis of specific proteins.

Transfer Process

• Proteins with negative charges migrate from the gel to the nitrocellulose membrane during the transfer process. After this step, antibodies can be applied to detect specific proteins.

Detection Methodology

• A secondary antibody, often enzyme-linked, is added which reacts with a substrate to produce a detectable precipitate at the protein location on the membrane.

Analyzing Results

• The presence of a band indicates that the target protein is present; absence suggests it is not. Molecular weight comparisons help identify and quantify proteins based on band intensity and thickness.

Significance of Western Blotting

• The term "Western blot" derives from its American origins, distinguishing it from similar techniques like Southern blot (DNA detection) and Northern blot (RNA detection).

Understanding Protein Localization

Example Analysis

• In an example with three samples, bands indicate where target proteins are present or absent. Band intensity provides insight into relative protein quantities across samples.

Limitations of Homogenates

• While homogenates reveal if proteins are present and their relative amounts, they do not provide information about protein localization within cells or tissues.

Subcellular Fractionation Techniques

Importance of Fractionation

• To determine protein localization within cells, subcellular fractionation must precede Western blotting. This involves separating cellular components through differential centrifugation.

Sample Preparation Steps

• Different cellular fractions such as nuclei, mitochondria, lysosomes, etc., can be analyzed separately in subsequent blots after initial homogenization.

Interpreting Subcellular Results

Case Study: Aromatase Detection

• In testing for aromatase across various fractions:

• Homogenate shows presence,

• Cytosol lacks detection,

• Microsomes show higher concentrations due to sample concentration during fractionation.

Conclusion on Distribution Insights

• Without subcellular fractionation prior to analysis, one cannot ascertain where within the homogenate a particular protein resides despite knowing its presence and quantity.

Example Application in Cancer Research

Ecadherin Expression in Tumors

Comparing Cadherin Levels in Samples

Understanding Cadherin and Control Comparisons

• The presence of cadherin allows for comparison across four samples, highlighting differences in intensity between those with and without cadherin.

• To calculate relative quantities, the intensity of cadherin bands is compared to a control band (e.g., actin), providing a ratio that indicates the relationship between cadherin and actin levels.

Techniques for Protein Detection

• Immunohistochemistry (IHC) and Western blotting are both based on antigen-antibody reactions but can face challenges in specificity.

• Non-specific antibody binding may result in multiple bands during Western blot analysis, indicating limitations in detecting specific proteins.

Fluorescent Proteins as an Alternative

Utilizing Fluorescent Proteins

• An alternative method involves using fluorescent proteins to study their distribution within tissues, which will be discussed later regarding DNA study techniques.

Immunohistochemistry vs. Western Blot

Key Differences Between Techniques

• IHC allows for localization of proteins within cells or tissues while providing relative quantification; however, it does not reveal protein location within sample components.

• Western blotting detects protein presence quantitatively but requires prior subcellular fractionation to determine protein distribution accurately.

Flow Cytometry: A Third Technique

Introduction to Flow Cytometry

• Flow cytometry is introduced as a technique that utilizes antigen-antibody reactions to separate and analyze cells rather than directly detecting proteins.

Process of Flow Cytometry

• In flow cytometry, tissue samples undergo immunohistochemistry with secondary antibodies tagged with fluorochromes to identify specific proteins in marked cells.

• Cells pass through a laser beam; fluorescence indicates the presence of fluorochromes while non-fluorescent cells allow light passage without emission.

Analyzing Cell Characteristics

Separation Based on Fluorescence

• Two detectors are used: one for fluorescence from tagged cells and another for light passing through all cells, allowing differentiation based on morphological characteristics.

Electromagnetic Separation

Flow Cytometry and Proteomics Techniques

Understanding Cell Morphology through Flow Cytometry

• The flow cytometer detects how light passes through cells, allowing analysis of cell granularity and size. This data is plotted on a graph to categorize cells based on morphological types.

• Blood cells are categorized by size and granule content; larger cells with more granules appear in one sector, while smaller ones with fewer granules occupy another.

• Flow cytometry utilizes antigen-antibody reactions from immunohistochemistry or immunofluorescence to differentiate fluorescent cells from non-fluorescent ones, aiding in the identification of cell types like granulocytes, monocytes, and lymphocytes.

• The technique provides insights into both the morphological characteristics of cells and the presence or absence of specific markers detected via immunofluorescence.

Techniques for Protein Analysis

• Previous discussions covered immunocytochemistry, Western blotting, and their roles in analyzing proteins within tissues. These methods provide information about protein distribution and relative quantities but are limited to small numbers of proteins.

• Proteomics encompasses techniques that analyze the total protein content within biological systems (cells, tissues, organs), offering a broader view than traditional methods.

Mass Spectrometry as a Foundation for Proteomics

• Mass spectrometry serves as the fundamental technique in proteomics. It analyzes proteins based on their mass-to-charge ratio after they have been ionized.

• The process begins with creating a homogenate containing proteins which are then digested into peptides using proteases. These peptides can be purified through various techniques such as chromatography or electrophoresis.

Analyzing Peptides Using Mass Spectrometry

• Ionized peptides are introduced into a mass spectrometer where they are separated based on their mass during radioactive emission exposure; smaller peptides move faster than larger ones.

• The resulting data allows for analysis along an axis representing mass-to-charge ratios. Peaks indicate relative quantities: taller peaks signify higher amounts of specific proteins while shorter peaks indicate lower amounts.

Identifying Protein Expression Patterns

• Data from mass spectrometry is analyzed using databases to identify expressed proteins within tissues and their relative quantities over time or between different tissue types.

Understanding Protein Expression Patterns

Importance of Protein Expression in Different Tissues

• The expression pattern of proteins varies across different tissues, highlighting the significance of differential gene expression during development.

• The final outcome of gene expression is the protein expression pattern, which provides insights into the types and quantities of proteins present in a specific tissue or cell at a given time.

Techniques for Analyzing Proteins

Steps in Proteomic Analysis

• Proteomics allows for the identification and quantification of proteins expressed by biological systems, focusing on both type and relative abundance.

• The analysis typically involves two main steps: separation (often through fragmentation) and mass spectrometry to analyze protein content.

Methods for Protein Separation

• While various methods exist for separating proteins, understanding that mass spectrometry can analyze total protein quantity and proportions is crucial.

• One effective technique for initial purification is two-dimensional electrophoresis, where proteins are separated first by pH and then by molecular weight.

Chromatography Techniques

Liquid Chromatography Overview

• Liquid chromatography separates proteins based on size; larger molecules take longer to pass through a column filled with porous beads.

• This method can also utilize charged beads to separate proteins based on their charge or be linked to antibodies for targeted retention.

Mass Spectrometry Process

• In mass spectrometry, samples are ionized using lasers, allowing smaller peptides to travel faster than heavier ones towards a detector.

• A graphical representation shows peaks corresponding to different peptides based on their velocity during detection.

Detailed Analysis Through Subcellular Fractionation

Insights from Subcellular Studies

• Subcellular fractionation enables the collection of peptides from various cellular compartments, providing detailed information about protein distribution within those fractions.

• For example, one study identified 22,260 peptides corresponding to 2,197 distinct proteins across subcellular locations.

Conclusion: Role of Proteomics in Biological Understanding

Summary of Proteomic Techniques

• Overall, proteomics facilitates an understanding of biological systems by analyzing expressed protein sets and their quantities through various purification techniques followed by mass spectrometry.

Techniques for Analyzing Proteins in Biological Systems

Overview of Protein Analysis Techniques

• The analysis of peptide segments leads to conclusions about the types and quantities of proteins present in a biological system.

• Two primary techniques discussed are immunocytochemistry and Western blotting, which utilize antigen-antibody reactions for protein analysis.

Immunocytochemistry vs. Western Blot

• Immunocytochemistry allows for the detection of protein distribution within tissues or cells, providing spatial context.

• In contrast, Western blotting detects the presence, relative quantity, and molecular weight of proteins in homogenates or plasma but does not reveal their distribution within tissues.

Flow Cytometry Insights

• Flow cytometry analyzes cells as they pass through a liquid flow and are assessed by laser light emission.

• This technique enables the separation of marked (fluorescently tagged) cells from unmarked ones based on their fluorescence characteristics.

Global Protein Analysis

• The concept of proteomics is introduced, allowing for a comprehensive analysis of all proteins expressed in a tissue at a specific time.

• While immunocytochemistry and Western blot focus on individual proteins, proteomics provides insights into the collective protein expression within biological systems.

Key Takeaways on Techniques

• Understanding the fundamentals, advantages, disadvantages, and applications of each technique is crucial for effective protein analysis.