BIOL240 Test 1 Review.pdf

BIOL240 – Written Assessment #1 Lecture 1: Introduction to Immunology A brief history of Immunology Edward Jenner, (1798) observed that milkmaids...

BIOL240 – Written Assessment #1 Lecture 1: Introduction to Immunology A brief history of Immunology Edward Jenner, (1798) observed that milkmaids who had previously caught cowpox did not later catch Smallpox.....Two months later, Jenner inoculated James with material from an individual with smallpox, with no effect, the boy was immune to smallpox. What is Immunology? The study of the immune system and its role in protecting the body against disease ® the molecules, cells, organs and systems responsible for the recognition and disposal of foreign (non- self) materials. Overview of Immune System Function Defense Against Foreign Entities: Protects against microbes, fungi, viruses, pathogens, and transplanted organs. Defense Against Abnormal "Self" Cells: Destroys abnormal or dead cells. Main Function Self / Non-Self Discrimination: Ability to distinguish between the body's own cells ("self") and foreign substances ("non-self") – invaders (like diseases) to helpful ones (like organ transplants) Immune System Dysfunction: A deficiency or dysfunction of the immune system can cause many disorders (immune mediated disorders: cancers, autoimmune diseases like type 1 diabetes, rheumatoid arthritis, lupus) Major Subdivisions of the Immune System Innate Immune System – 1st line of defense Non-specific Immunity (antigen nonspecific): Doesn’t target specific pathogens (antigens). Instead, recognizes general features common to many invaders (e.g., bacteria or viruses), responding the same way to a range of threats. Immediate Maximal Response (antigen-independent): Immediate and does not require prior exposure to the pathogen. Acts as soon as threat is detected, without needing time to adapt. Exposure Results in No Immunological Memory: Does not remember past encounters with pathogens. Each time the body is exposed to the same threat, the response is identical. Includes: anatomical barriers, cellular components, humoral components (secretory molecules) Anatomical Barriers Mechanical Factors Chemical Factors Biological Factors ® Physical – epithelial surfaces ® Fatty acids – sweat ® Normal flora – skin, GI tract (gut ® Movement of cilia, peristalsis ® Lysozyme – tears, saliva, nasal secretions microbiome) ® Washing – tears, saliva, urine ® Low pH – sweat glands, gastric juices ® Trapping effect of mucus Cellular Components ® Neutrophils, Eosinophils, Basophils ® Mast cells, Monocytes – Macrophages, Dendritic cell ® Natural Killer cells Humoral Components ® Opsonization (coating pathogens to enhance phagocytosis). Complement System: Series of proteins that contribute to: ® Inflammation. ® Cytolysis (breaking down cells) Cytokines: Chemical messengers affecting other cells, including: Interleukins, Colony Stimulating Factors. Tumor Necrosis Factors, Interferons  Adaptive Immune System – 2nd line of defense Specific Immunity (antigen-specific): Recognizes and responds to specific pathogens or antigens to specialize a response for a more effective defense. Delayed Response (antigen-dependent): Takes time to mount a full response after initial exposure to a pathogen because immune cells need to recognize the antigen, activate, and proliferate before effectively attacking the pathogen Immunological Memory: Creates memory cells that "remember" the specific antigens of that pathogen – when exposed to the same pathogen again, it can respond more quickly, often neutralizing the threat before it can cause harm. Includes: secretory molecules (humoral components) and cellular components. Cellular Components T lymphocytes (T-Cells) B lymphocytes (B-Cells) Helpers (communicate) Memory (antigen specific) Cytotoxic (destroy cells) Plasma (antibodies) Humoral Components Antibodies (Immunoglobulins): Cytokines: Glycoproteins produced by B lymphocytes in response to foreign Same as in the innate system, act as messengers to influence substances. other immune cells. Cellular Components vs. Humoral Components Cells à part of an organ or free in circulation. Humoral components à free in circulation. Innate Immune Response – Recognition of Pathogens: The innate immune system uses Pathogen Recognition Receptors (PRRs) to detect pathogens. PRRs are found on the cell surface (for extracellular pathogens) and inside the cell (for intracellular pathogens). A key type of PRR is Toll-like receptors (TLRs), which recognize pathogen structures known as Pathogen-Associated Molecular Patterns (PAMPs). Adaptive Immune Response – Recognition of Pathogens: The adaptive immune system targets specific pathogens. Lymphocytes are responsible for the specificity, diversity, and memory in adaptive immunity. T cells have T cell receptors (TCRs) that recognize specific antigens and activate cell- mediated immunity. B cells have B cell receptors (BCRs) that recognize specific antigens and activate humoral immunit  Lecture 2: Antigens and Antibodies Factors influencing the Immune Response Two Main Influences on Immune Response 1.) Human Biological Properties 2.) Traits of Immunogens 1.) Human Biological Properties: These properties affect the nature of an individual's immune response: § Age: Immune responses can vary with age, typically being stronger in childhood and gradually declining in older age. § Overall Health: Including the presence of chronic illnesses or infections, can impact their immune function. § Dose: The amount of the immunogen (antigen) encountered can influence the strength and duration of the immune response 2.) Traits of Immunogens (Immunogenicity): The ability of an immunogen to stimulate an immune response depends on several factors: § Macromolecular Size: Immunogens are more likely to provoke a strong response if they are larger than 10,000 Daltons. § Chemical Composition and Molecular Complexity: ® Proteins: Generally, the most immunogenic due to their size and complexity. ® Carbohydrates: Can also be immunogenic, especially when complex. ® Lipids: Typically, less immunogenic unless attached to proteins (lipoproteins). § Degree of Foreignness: The greater the difference between the immunogen and the host's molecules, the stronger the immune response. More "foreign" substances are better recognized and targeted by the immune system. Definitions: Immunogens: Substances that can provoke an immune response (A piece of the immunogen can be an antigen) Antigens: Molecules that can bind/react to specific antibodies or immune receptors; all immunogens are antigens, but not all antigens are immunogens. Haptens: Small molecules that become immunogenic only when attached to larger carrier proteins. Epitopes: Specific parts of an antigen that are recognized by antibodies or immune cells which induces the immune response (antigenic determinant or determinant site) Immunoglobulins (Ig): Glycoprotein molecules that are produced by plasma cells in response to an immunogen (antigen à epitope) and which function as antibodies Antibody Synthesis Antigen encountered à (B cells) recognize the antigen as a non-self à immune response. ® Some antigens do not elicit an immune response. Production of antibodies is induced when the host’s lymphocytes come into contact with a foreign antigenic substance that binds it its receptor (BCR) – triggers activation and proliferation of the cell Immune Response Primary Immune Response Secondary, Memory or Anamnestic Antibody Response Antibody (Ab) production proceeds in the following 4 phases: Repeat exposure to the same antigenic stimulus produces an antibody response that exhibits the same 4 phases but Lag Phase with different: No antibody detected, lasts 5-7 days. § Time (Shorter lag phase) Log Phase § Antibody Titer (Concentration (titer) is higher) Antibody concentration increase exponentially. Plateau Phase Antibody production and decay are equal. Decline Phase Antibody decay is greater than antibody production.  Structure Although each class of immunoglobulin has unique properties, all immunoglobulins molecules share many common features. § Polypeptide chains; 2 identical heavy (H) chains, 2 identical light (L)chains § Variable (V) Region: Region responsible for antigen specificity. § Constant (C) Region: Determines the class and effector function. § Hinge Region: Provides flexibility to the antibody. § Disulfide Bonds: Link chains and provide structural stability. § Fc (Fragment Crystallizable) Region: Mediates effector functions. § Carbohydrate Region: Contributes to stability and activity. § Fab (Fragment Antigen Binding) Region: Contains the antigen-binding sites. Function Antigen Binding: Each immunoglobulin is specific to a particular antigenic determinant. Binding to the antigen is the primary function of antibodies, which can help protect the host from pathogens. Effector Functions: § Opsonization: Coating pathogens (antigens) to enhance phagocytosis. § Complement activation: Initiating the complement cascade to enhance the immune response § Placental transfer: Transfer of antibodies from mother to fetus (specific to certain classes). Classes Location: Functions: Found in intravascular and extravascular § Provides immunity to the fetus or newborn. IgG (Gamma spaces. § Fixes complement (activates the complement Heavy Chains) cascade). § Opsonizes pathogens for phagocytosis. § Neutralizes toxins. Location: Functions: IgM (Mu Heavy Found in intravascular spaces and on the § Fixes complement. Chains) surface of B cells (as B cell receptor, BCR). § Opsonizes pathogens for phagocytosis. § Neutralizes toxins Location: Function: IgA (Alpha Heavy Primarily found in secretions such as tears, § Protects mucous membranes Chains) saliva, intestinal fluids, breast milk, and sweat. Location: Functions: IgD (Delta Heavy Found on the surface of B cells (as BCR). § Primarily found in plasma; role in B cell activation is Chains) less understood. Location: Functions: IgE (Epsilon Found in circulation. § Mediates hypersensitivity (allergic) reactions. Heavy Chains) § Responsible for immunity to parasites. Bonding Affinity: The bond strength between a single antigenic determinant (epitope) and a single combining site of the antibody. ® Each antibody has at least two identical antigen-binding sites, giving it a specific valence (the number of antigen-binding sites). Avidity: The overall strength with which a multivalent antibody binds to a multivalent antigen. ® Increased when a multivalent antigen interacts with multiple antibody binding sites, significantly enhancing bonding strength. As the epitope and antigen-binding site come close together, several types of non-covalent bonds hold them together, including: § Ionic Bonds § Hydrophobic Bonds § Hydrogen Bonds § Van der Waals Forces Goodness of Fit § The strongest bonding occurs when the shape of the antigenic determinant and the antibody's antigen-binding site conform well to each other. § A good fit allows for multiple simultaneous non-covalent bonds, minimizing the chances of bond disruption. Cross Reactivity Refers to an individual antibody's ability to react with more than one antigenic determinant, highlighting the versatility of antibodies in recognizing similar structures.  Lecture 3: The Complement System Complement System Activation & Functions Part of the innate immune system, that enhances the ability of antibodies and phagocytic cells to: ® Clear microbes and damaged cells from an organism ® Promotes inflammation ® Attack a pathogen’s cell membrane...can be activated by the adaptive immune system § Consists of small proteins found in body fluids and tissues, synthesized by the liver and circulate as inactive precursors (zymogens). - Activated by proteolytic cleavage - At site of infection à activated locally and trigger a series of potent inflammatory events Activation: through a triggered-enzyme cascade Cascade: an active complement protein (enzyme = serine protease) generated by cleavage of its zymogen precursor à cleaves its substrate, (another complement zymogen), to its active enzymatic form (serine protease). - this in turn cleaves and activates the next zymogen in the complement pathway active complement protein à substrate (zymogen) à active complement protein à substrate à active complement protein à substrate à active complement protein à substrate à active complement protein à Three Pathways for Activation These pathways are triggered by different molecules but ultimately lead to the same effects: § Produce complement proteins that coat pathogens, making them easier for phagocytes to engulf (Opsonization) § Small fragments of some complement proteins attract and activate more phagocytes to the site of infection (Inflammation) § Form pores in bacterial membranes, leading to their destruction (Cytolysis/MAC) Classical Pathway MB-Lectin Pathway Alternative Pathway Antigen: Antibody Complexes Lectin binding to pathogen surfaces Pathogen surfaces Complement Activation Recruitment of inflammatory cells Opsonization of pathogens Killing of pathogens  The Classical Pathway You will be given a diagram of the classical pathway on the test. You will still need to know how the other pathways function and interconnect with the classical pathway. § C1qrs complex binds to the pathogen, held together by calcium § When two or more C1q heads attach to antibodies on the pathogen, it changes the shape of the C1 complex § This activates C1r, turning it into a serine protease, which then activates C1s § C1s, now a serine protease, can cleave and activate C4 and C2 proteins, continuing the complement cascade. Step 1: § C1s cleaves C4 into C4a (floats off, causes inflammation) and C4b (binds to pathogen). § C1s cleaves C2 into C2a (binds to pathogen) and C2b (floats off, gets degraded). § C4b and C2a combine to form C3 convertase (C4b2a). Step 2: § C3 convertase cleaves C3 into C3a (floats off, causes inflammation) and C3b (binds to pathogen). § C3b joins C4b2a to form C5 convertase (C4b2a3b), and C3b helps phagocytes engulf the pathogen. Step 3: § C5 convertase cleaves C5 into C5a (floats off, causes inflammation) and C5b (binds to pathogen, starting the Membrane Attack Complex - MAC). § C5b binds to the pathogen. § Then C6, C7, C8, and C9 bind to form the MAC (Membrane Attack Complex) The MAC damages the pathogen by: § Forming channels (holes) in the membrane. § Rearranging the membrane molecules. This causes: § Water to flow into the cell § Loss of electrolytes from the cell. § The pathogen is destroyed as a result. Amplification Loops: C4a, C3a, C5a: Inflammatory Response: § 1 C1 bound to an antibody can § C3a and C5a attract immune cells to § Inflammation isolates the activate 30 C4 molecules. the infection (neutrophils, monocytes, infection or injury, allowing § 1 C3 convertase can activate 200 C3 etc.). immune cells to clear damaged molecules. § C4a, C3a, C5a trigger inflammation cells and microbes from the area. The Lectin Pathway by releasing histamine and heparin The Lectin Pathway uses a protein similar to C1q called mannose-binding lectin (MBL) to start the complement cascade. from mast cells and basophils, leading § MBL is a protein in the blood that to muscle increases contraction during and and binds to mannose (a sugar inflammation increased blood vessel found on bacteria, yeast, viruses, and parasites). permeability. MBL Structure: § Has 6 globular heads. § Contains 3 serine proteases: MASP-1, MASP-2, and MASP-3 (MASP = mannose-associated serine proteases). When MBL binds to a pathogen: § MASP-1 and MASP-2 become active and cleave: ® C4 into C4a and C4b. ® C2 into C2a and C2b. From here, the pathway follows the same steps as the Classical Pathway.  The Alternative Pathway The Alternative Pathway can start on any microbial surface without needing a specific pathogen- binding protein. § It begins with the spontaneous breakdown of C3, creating C3a and C3b. § C3b binds to the pathogen or host cell. This pathway also includes: factors B, D, & P The Alternative Pathway Steps: 1. C3 is cleaved into C3a (anaphylatoxin) and C3b. C3b binds to factor B, and together they attach to the cell surface. 2. Factor D cleaves factor B into Ba and Bb. Bb combines with C3b to form C3bBb, which acts as C3 convertase. 3. More C3 is cleaved by C3 convertase, creating a loop that produces more C3bBb. Factor P (properdin) stabilizes this enzyme. 4. When another C3b joins the complex, it becomes capable of cleaving C5. This forms the C5 convertase (C3bBbPC3b). 5. After C5 is cleaved, the pathway continues just like the Classical Pathway. This pathway amplifies the immune response and ultimately leads to the formation of the Membrane Attack Complex (MAC). All pathways (Classical, Lectin, Alternative) meet at the cleavage of C3 into C3a and C3b. The final stage is the same for all pathways. Main functions of complement: inflammation, phagocytosis, and creating the Membrane Attack Complex (MAC). Other Key Functions of Complement Removal of Immune Complexes: § Antibodies form large antigen-antibody complexes. § C3b breaks them down into smaller pieces, allowing macrophages to clear them Removal of Necrotic Cells § Necrotic (dying) cells activate complement, leading to C4b and C3b on the cell surface. § These cells are cleared by phagocytes. § Clinical Consequences: In conditions like ischemia (lack of blood flow), complement activation can damage healthy surrounding tissue. Responses to Viruses: § Complement helps defend against viral infections like HIV and influenza by opsonizing and lysing them for phagocytosis.  Lecture 4: Cellular Components Primary and Secondary Lymphoid Organs The immune system is localized in several parts of the body. § Immune cells develop in the primary § Immune responses occur in the organs: bone marrow & thymus. secondary organs. Primary Lymphoid Organs (development of immune cells) Produces hematopoietic stem cells. Bone Marrow: Maturation of B cells and NK cells. Thymus: Maturation of T cells. Secondary Lymphoid Organs (immune response occurs) Sites for contact with antigens, increasing immune response probability Includes: § Spleen § Lymph nodes § Mucosal associated lymphoid tissue (MALT) § Cutaneous associated lymphoid tissue (CALT) Vessels run alongside the circulatory system and flow towards the heart using skeletal muscle contractions. (No pump required) Spleen: Filters blood for microbes and toxins Lymph Nodes: Filters lymph before it returns to blood Both contain macrophages and T cells for phagocytosis and immune response initiation Cells of the Innate Immune System: § Neutrophils § Monocytes § Basophils § Macrophages § Mast cells § Dendritic cells § Eosinophils § Natural Killer cells § Are a population of short-lived cells Granulocytes or Function: Polymorphonuclear (PMN) Leukocytes § Phagocytosis – enzyme rich lysosomes facilitate destruction of infectious (basophils, mast cells, eosinophils and microorganisms neutrophils) § Protection against infection. § Are phagocytic cells § Monocytes migrate and differentiate into macrophages in the central nervous tissue Function: Monocytes & Macrophages § Phagocytosis – to engulf and break down trapped materials into simple amino acids, sugars for reuse or excretion § Antigen Presentation – take up antigens, process them by denaturation or partial digestion and present the fragments to antigen-specific T cells § Reside in the tissues: skin, mucous membranes, heart, lungs, liver, kidneys Function: Dendritic cells § Phagocytosis – to engulf and break down trapped materials into simple amino acids, sugars for reuse or excretion § Antigen Presentation – take up antigens, process them by denaturation or partial digestion and present the fragments to antigen-specific T cells ,  Phagocytosis 1.) Adherence: Phagocytes bind to pathogens using Neutrophils are attracted to the site of infection – opsonins. chemotaxis Adherence – physical contact between the Neutrophils are able to leave the vessels and enter tissue phagocytic cell and the microorganisms – diapedesis (pathogen) Macrophages & dendritic cells are already in the tissue. Opsonins – Serum proteins that act as Pathogen recognition receptors on these cells recognize (markers / tags) on pathogen to help with PAMPs and bind to them adherence 2.) Engulfment: outflowing of cytoplasm to surround the Pseudopodia (extensions) start to encase pathogen. microorganism. 3.) Phagosome Formation: microorganism is completely Complete vacuole formed around pathogen that carries it surrounded by a part of the cell membrane. toward the center of the cell. 4.) Granule Contact: lysosomal granules contact and fuse Little pouches filled with various enzymes start to bind to with the phagosome. the vacuole containing the pathogen 5.) Phagolysosome Formation: contents of the lysosome Enzymes start to digest the pathogen. are emptied into the membrane space. 6.) Digestion: Pathogen is broken down by enzymes. 7.) Excretion: contents of the phagolysosome are expelled Pieces of the pathogen are released through tiny to the outside of the cell by exocytosis. vacuoles Antigen Presentation § Pathogen fragments (degraded proteins) are transported to the cell's surface where T cells can recognize them. § Cells use MHC class I and MHC class II molecules to present antigens to other cells. Major Histocompatibility Complex molecules – cell surface molecules ® Also known as Human Leukocyte Antigens class I and II Function: § Bind to peptides produced when proteins are broken down inside the host cell. § Present these peptides to T cells. § MHC molecules "sample" the inside of the host cell, helping T cells determine if the cell is infected or contains a foreign component. Antigen Presenting Cells (APCs): § APCs ingest a microorganism and break down its antigens into small peptides. § These peptides combine with MHC Class II molecules and are displayed on the APC's surface. This process is called exogenous processing. NK Cell Function § Are large granular lymphocytes that are cytotoxic. Function: Natural Killer Cells § Recognize altered membranes of abnormal cells destroy them Lack specificity in their response à essential to their function as an early defender against pathogens. (2) kinds of receptors on their surface: 1. Inhibitory receptor – if the inhibitory receptor receives a signal – no response 2. Activating receptor – if an activating receptor receives a signal – cell is destroyed. Immunological Tools and Techniques Immunoprecipitation (IP) Purpose: To isolate small amounts of a protein of interest from a heterogeneous cell or tissue extract using a target-specific antibody. IPs are performed to investigate the: ® Identity ® Activation ® Structure ® Modification state ® Expression …of a protein of interest 3 Main Steps: 1. Incubate resin (beads) and target-antibody with a sample that contains your protein of interest Beads (resin): Provides the “support” or surface for antibodies to bind § Typically coated in Protein A or Protein G or Protein A/G § Can be magnetic or agarose Target-antibody: Selected to target your protein of interest § Generated by inoculating an animal with protein of interest (polyclonal) OR generating a hybridoma (monoclonal) § Antibodies will stick to beads because of Protein A or G Sample (heterogenous): Tissue culture cells § Tissue obtained from patients or animals (heart, liver, etc) § Blood samples (RBC, immune cells, platelets, etc.) Incubating beads and antibody with sample containing protein of interest § Method 1: Pre-immobilized antibody approach Method 2: Free antibody approach 2. Separate the bead/antibody/protein complex from the rest of the sample (ie. the precipitate) § Centrifugation + wash steps § Magnet + wash steps 3. Analysis § Western Blot to look at protein of interest ® Is it present? ® Has it been activated (phosphorylation) § Enzyme activity assays § Mass spectrometry § Many more! Co-Immunoprecipitations (co-IP) Purpose: What proteins are interacting with my protein of interest? § Proteins in cells typically have many interacting partners. By immunoprecipitating one protein, you may “pull-down” interacting partners.  ELISAs (Enzyme-Linked Immunosorbent Assays) Purpose: Used to detect (qualitative) and measure (quantitative) proteins of interest in biological samples 3 Main Steps: 1. Coating (with either antigen or antibody) Coat a polystyrene plate (typically 96-well) with antigen 2. Blocking (typically with the addition of bovine serum albumin [BSA]) Blocking (BSA, ovalbumin, aprotinin, or other animal proteins) - Incubate - Wash This step minimizes false positive results 3. Detection Detection antibody - Incubate - Wash Antibody is conjugated with an enzyme (HRP or AP) A substrate is added (hydrogen peroxide, or PNPP). ELISAs are typically colorimetric 4. Final read Types of ELISAs Type Key Points Advantages Disadvantages § Only one antibody is used, § Low sensitivity Binds antigens, including desired target, in a sample so cross-reactivity is not a directly to the plate. An enzyme-conjugated antibody concern § Non-specific binding of is then added as a probe for the desired analyte antigens so background may § Rapid be high § There is a risk of antibody Binds antigens, including the desired target, in the cross-reactivity sample to the plate. However, it involves two § High sensitivity antibodies; a primary antibody and a secondary § Non-specific binding of conjugated antibody. sample antigens so background may be high The target is bound between a capture antibody and § Choosing the right antibody the conjugated detecting antibody, creating a § Highly sensitive and specific pair can be time-consuming “sandwich” § Rapid Involves competition between the binding of the § Requires little/ no sample sample antigen and conjugated antigen to a specific pre-processing amount of antibody. The more antigen in the sample, § Low specificity the less conjugated antigen binds and the lower the assay signal. § Useful for small targets that cannot easily be bound with two antibodies Enzyme-linked conjugated antibody: An antibody that is chemically linked to an enzyme. This conjugate is used in assays to produce a measurable signal, typically through a color change when the substrate is added. Primary antibody: An antibody that specifically binds to a target antigen. It is the first antibody used in an assay to detect or quantify the antigen of interest. Secondary antibody: An antibody that binds to the primary antibody. It is often conjugated to an enzyme or a fluorescent marker, enhancing the signal for detection. Capture antibody: An antibody used to immobilize the target antigen on a solid surface (such as a plate) in an assay. It captures the antigen from a sample for further detection.  Western Blotting Purpose: used to detect specific protein molecules from among a mixture of proteins. Western blots can also be used to evaluate: ® size of a protein of interest ® measure the amount of protein expression Western Blotting – Overview 1.) Sample Preparation: Lyse the sample in an appreciate lysis buffer. Reduce and denature the sample with sample buffer containing SDS and β-mercaptoethanol. Heat the sample at 95˚C 5 mins. 2.) Gel Electrophoresis: Load an optimized quantity of sample onto the gel. Perform gel at 100-200V 30 mins. Proteins separate according to size with smaller proteins migrating more quickly through the gel. 3.) Membrane Transfer: Prepare cold transfer buffer and the membrane. Assemble the transfer sandwich with the gel near the (-) cathode and the membrane near the (+) cathode. The negatively charged proteins will migrate out of the gel onto the membrane. Perform transfer 30V o/n @ 4˚C or 100V for 1hr. 4.) Immunodetection: Use a total protein stain to check the transfer efficiency. Block the membrane in an appropriate blocking buffer (milk, BSA, etc). Incubate the membrane with primary antibody 1 hr RT or 4˚C o/n. Incubate with secondary antibody 1 hr RT. Perform detection step (ex. chemiluminescent substrate kit). Perform data analysis. Membrane Transfer: § Once your protein samples are run on an SDS-PAGE gel, you can transfer the proteins to a membrane (typically nitrocellulose, polyvinylidene difluoride (PVDF)) § Transfer is performed in a buffer (cold) with an electrical current flowing through Blocking Step: Typically BSA or milk in buffer solution (like TBS-T) § Important to prevent non-specific binding of antibodies to the membrane, enhancing the signal-to- noise ratio and improving the sensitivity and consistency of the results. Wash Steps: Western blotting consists of a series of incubations with different immunochemical reagents separated by wash steps. § Necessary to remove unbound reagents and reduce background, increasing the signal-to-noise ratio. § Insufficient washing may result in high background § Excessive washing may result in decreased sensitivity caused by elution of the antibody and/or antigen from the blot. § Common wash buffers: Tris-buffered saline (TBS) & Phosphate buffered saline (PBS) Antibody Detection What are antibodies? - Part of animal immune response - Utilized by scientists to detect proteins of interest How do antibodies work? - Detection of antigens 2 types of antibodies are required: 1.) Primary (1˚) Antibody 2.) Secondary (2˚) Antibody  Detection Methods: Colorimetric Detection: Chemiluminescence Detection: Fluorescence Detection: Secondary antibody is bound to an Secondary antibody is bound to an Secondary antibody is labeled with enzyme. enzyme. a fluorophore. § When substrate is added (your § When substrate is added (your § A light source excites the detection reagent), a colored detection reagent), a light is fluorophore and the emitted precipitate will accumulate on the produce that can be detected fluorescent signal is captured by blot using X-ray film or by a charge- a camera to produce the final coupled device (CCD) imager image Pros: Easy to use & inexpensive, can Pros: High sensitivity, quick (short Pros: Large dynamic range, reprobe same membrane with different exposure times) multiplexing, stable (can store colors Cons: X-ray film $, trial and error labelled blots for weeks and image Cons: medium sensitivity during exposure again) Cons: cost #