Notes 2: Analysis of Gene Expression (protein level)

immunofluorescence, fusion tagging, affinity binding

Fluorescence Microscopy Labeling Techniques:

• ________

• _________ _________

• __________ ______

specific

Production of Polyclonal Antibodies:

• Selection of Antigen:

  • Choose a ____ antigen of interest, such as collagen alpha one, for antibody production

purify, inject, different

Production of Polyclonal Antibodies:

• Animal Immunization:

  • ___ the antigen, such as collagen, and ___ it into an animal (e.g., rabbit, goat, donkey).

  • Select an antigen from a species ___ from the immunized animal to avoid self-tolerance.

antibodies

Production of Polyclonal Antibodies:

Immune Response:

• The injected antigen triggers an immune response in the animal.

• B cells are activated, leading to the production of _____ against various epitopes of the antigen

epitopes, epitope, polyclonal

Production of Polyclonal Antibodies:

• Polyclonal Antibodies:

  • Multiple ______ of the antigen are recognized by different clones of B cells.

  • Each clone produces antibodies specific to a particular ___.

  • Resulting antibodies constitute a ____ antibody mixture.

plasma

Production of Polyclonal Antibodies:

• Memory B Cells and Plasma Cells:

  • Selected B cell clones expand and differentiate into memory B cells and plasma cells.

  • Memory B cells provide long-term immunity, while ___ cells secrete antibodies.

blood

Production of Polyclonal Antibodies:

• Antibody Purification:

  • After a few weeks, collect ___ from the immunized animal and purify antibodies from the serum.

  • Affinity purification techniques, such as using a collagen-bound column, can isolate antibodies specific to the antigen of interest.

epitopes, epitope

Production of Polyclonal Antibodies:

• Polyclonal Nature:

  • Polyclonal antibodies recognize multiple _____ of the antigen.

  • Each clone of B cells produces antibodies that recognize a specific ___, resulting in a diverse antibody population.

Inject

Production of Monoclonal Antibodies:

• Animal Selection and Antigen Injection:

  • Typically use mice, although rabbits and rats are also used.

  • ___ the antigen into the chosen animal to trigger an immune response.

spleen, myeloma, hybridoma

Production of Monoclonal Antibodies:

• Spleen Cell Fusion:

  • Obtain ___ cells from the immunized animal.

  • Fuse spleen cells with ___ cells (B cell lineage) to create hybridoma cells.

  • ___ cells will produce antibodies specific to the antigen.

hybridoma, antibody

Production of Monoclonal Antibodies:

• Clonal Selection and Expansion:

  • Select fused _____ cells and expand them in culture.

  • Each hybridoma cell represents a specific B cell clone producing a unique ____.

individually, epitope, cross-react

Production of Monoclonal Antibodies:

• Monoclonal Antibody Screening:

  • Plate hybridoma cells _____ and expand them.

  • Induce cells to produce and secrete antibodies.

  • Screen antibodies to identify those specifically recognizing the desired antigen _____.

  • Ensure antibodies do not ___-__ with similar proteins.

single, large

Production of Monoclonal Antibodies:

• Advantages of Monoclonal Antibodies:

  • Highly specific for a ___ epitope.

  • ____ amounts of pure antibodies can be obtained.

  • Suitable for therapeutic applications due to purity and specificity.

GMP

Production of Monoclonal Antibodies:

• Manufacturing Pipeline:

  • Monoclonal antibodies produced under Good Manufacturing Practice (___) standards.

  • Ensures cleanliness and absence of contaminants for safe human use.

  • Limited variability between batches compared to polyclonal antibodies.

epitope, signal

Disadvantages of Monoclonal Antibodies:

• Lower Signal Intensity:

  • Detection limited to a single ___, resulting in only one antibody binding per protein copy.

  • Each antibody produces one quantum of signal, potentially leading to lower overall __ compared to polyclonal antibodies.

broader

Disadvantages of Monoclonal Antibodies:

• Limited Versatility:

  • Monoclonal antibodies may not always be suitable for certain applications where ____ recognition is desired.

  • In cases where cross-reactivity is needed, monoclonal antibodies may not provide the desired outcome.

longer, expensive

Disadvantages of Monoclonal Antibodies:

• Development Complexity:

  • Development process for monoclonal antibodies is ___ and more ___ compared to polyclonal antibodies.

  • Requires extensive screening and optimization to identify antibodies with desired specificity and affinity.

antibodies, antigens

Immunoassays Overview:

• Principle:

  • Utilizes the strong interaction between ___ and ___.

  • Mixtures of molecules (e.g., proteins, tissue extracts, serum) are analyzed, with the target molecule of interest (antigen) being recognized by specific antibodies.

primary, washing, antigen

Immunoassays

• Procedure:

  • Antibodies (__) that selectively bind to the antigen of interest are added to the mixture.

  • After incubation, non-specifically bound molecules are removed through ___.

  • The remaining molecules bound to the antibodies are those containing the ___ of interest.

directly

Immunoassays

Detection Methods:

• Direct Conjugation:

  • Primary antibodies are ___ conjugated to fluorophores or chromogens for visualization.

  • Signal detection can occur under epifluorescent or confocal microscopy, Western blotting imagers, or plate readers.

constant

Immunoassay

• Detection Method: Secondary Antibodies:

  • If primary antibodies are not directly conjugated, secondary antibodies are used.

  • Secondary antibodies recognize and bind to the ___ fragment of primary antibodies.

  • Secondary antibodies are conjugated to fluorophores or chromogens.

polyclonal

Immunoassays

• Secondary Antibodies:

  • Secondary antibodies are typically _____ and recognize various regions within the constant fragment of primary antibodies.

  • They amplify the signal by binding to multiple primary antibodies, enhancing detection sensitivity.

species

Immunoassays

• Species Specificity:

  • Constant fragments of antibodies are ____-specific.

  • Anti-species secondary antibodies are generated by immunizing animals with constant fragments from a specific species.

  • This allows for recognition of primary antibodies from different species, facilitating detection in a wide range of immunoassays.

dead, fixed, permeabilized

Immunoassays: Specimen Preparation

• Tagging Method:

  • Utilizes antibodies conjugated to fluorophores or chromogens for detection.

  • Requires the specimen to be __, __, and potentially ___, depending on the target location within the cell.

amphipathic, detergents

Immunoassays

• Permeabilization Process:

  • Necessary for targeting intracellular components.

  • Achieved by using detergents, which are ____ molecules containing both hydrophobic and hydrophilic regions.

  • _____ create pores in the plasma membrane, allowing antibodies to penetrate into the cell.

antibodies

• Purpose:

  • Permeabilization enables the entry of ____ into the cell, facilitating the detection of intracellular antigens.

  • Without permeabilization, antibodies cannot diffuse through the intact plasma membrane.

fluorescent, target, live

• Fusion tagging involves genetically fusing a ______ protein, such as green fluorescent protein (GFP), with the ______ protein of interest.

• Enables visualization of the target protein in ___ cells without the need for fixation or permeabilization.

process

Fusion Tag Proteins

• ___:

  • Obtain the cDNA (coding DNA) of the protein of interest.

  • Construct a fusion protein by ligating the GFP (Green Fluorescent Protein) cDNA to the cDNA of the protein of interest.

  • Decide the attachment site (N-terminus, C-terminus, or internally) based on experimental needs.

  • Transfect the fusion protein construct into cells.

linker, fold

• Fusion proteins typically include a ___ region between GFP and the protein of interest to ensure proper folding.

• Linker allows the protein of interest to __ normally without hindrance from GFP.

Fusion tagging

• ___ ___ enables real-time visualization of protein localization and dynamics within living cells.

fluorescence

• The last way to label fluorescently is to use affinity binding

• so using dyes that instead of being chromogenic, like the ones used in histology, instead of producing color, produce _____.

intercalating, DNA, DNA

• DAPI (4',6-diamidino-2-phenylindole):

  • A DNA ______ dye that fits between the two helices of the DNA double helix.

  • Normally non-fluorescent, but becomes fluorescent when bound to ___.

  • Used to visualize ___ in cells.

  • Example: Visualizing individual chromosomes during cell division (mitosis), highlighting the mitotic spindle.

mushrooms, polymerization, F-actin, actin

• Phalloidin:

  • A toxin derived from poisonous ___.

  • Binds to F-actin and inhibits ___.

  • Used to visualize actin filaments (__-__) in cells.

  • Fluorescently labeled phalloidin enables visualization of ___ filaments.

  • Note: Phalloidin is toxic to cells and can lead to cell death shortly after staining.

snake, acetylcholine receptors, acetylcholine

• Bungarotoxin:

  • Derived from ____ venom.

  • Binds to _________ ______ at the neuromuscular junction.

  • Blocks the flow of _____, inhibiting muscle contraction.

  • Used to label neuromuscular junctions in conjunction with fluorescent markers.

  • Example: Labeling axons and neuromuscular junctions using thy1 GFP (green fluorescent protein) and bungarotoxin conjugated to Alexa Fluor 594.