BS2092 Molecular Cell Biology: Antibodies and Immunofluorescence Microscopy

BS2092 Molecular Cell Biology: Antibodies and Immunofluorescence Microscopy

Introduction

  • Course Details: BS2092 Molecular Cell Biology, Dr. Raj Patel, School of Biological and Biomedical Sciences
  • Contact: rp31@le.ac.uk
  • Lecture Topic: Antibodies and Immunofluorescence Microscopy

Antibody Structure

  • Definition: Antibodies are proteins produced by the vertebrate immune system as a defense against infection.
  • Structure: A typical antibody molecule comprises:
    • Variable region – responsible for antigen binding
    • Constant region – determines the class of the antibody

Types of Antibodies

Antigenic Determinants (Epitopes)

  • Epitopes are specific parts of the antigen recognized by antibodies. They can be either linear or conformational.

Polyclonal Antibodies (PAb's)

  • Definition: Antibodies that recognize multiple epitopes on an antigen.
  • Production:
    • Inexpensive to produce
    • Skills required for production are low
    • Relatively quick to produce
    • Can generate non-specific antibodies
    • Characterized by batch-to-batch variability.

Monoclonal Antibodies (MAb's)

  • Definition: Antibodies that recognize one specific epitope on an antigen.
  • Production:
    • More expensive to produce
    • Requires training in hybridoma technology
    • Takes a relatively long time to produce
    • Once established, they are a constant and renewable source of antibodies
    • Characterized by low batch-to-batch variability.

Fluorescent Antibodies

Fluorophore

  • The term used for a molecule that can emit light upon excitation, often used in conjunction with antibodies for visualization.

Fluorescence Microscopy

What is Fluorescence?

  • Definition: Fluorescence is a process where certain molecules (fluorophores) absorb light at one wavelength and emit it at a longer wavelength.
  • Excitation and Emission Process:
    1. Molecules are excited from ground state to an excited state.
    2. They then return to the ground state, emitting light in the process.
    3. Some energy loss occurs due to conformational changes and interactions with the molecular environment.

Characteristics of Fluorescent Dyes

  • Each fluorescent dye has a unique excitation and emission spectrum.
  • Example of a common dye: FITC (Fluorescein Isothiocyanate)
    • Excitation: 494 nm
    • Emission: 519 nm
  • Fluorescent dyes play a critical role in immunofluorescence microscopy to detect protein localization.

Types of Fluorescent Dyes Used in Cell Biology

  1. Small Fluorescent Dyes:
    • Examples include FITC and TRITC, commonly used for staining.
  2. Fluorescent Proteins:
    • GFP (Green Fluorescent Protein) and its variants (RFP, YFP) are used as markers to study protein localization in live cells.
  3. Dyes for Specific Organelles:
    • Dyes like Mitotracker for mitochondria and DAPI for DNA.
  4. Dyes for Monitoring Ions:
    • e.g., Fura-2 or Calcium Green for monitoring intracellular Ca2+ ions.
  5. Dyes for Measuring pH:
    • Fluorescent dyes can also measure the intracellular pH in live cells.

Fluorescence Microscope Configuration

  • Components:
    1. First Barrier Filter: Passes blue light with wavelengths of 450-490 nm.
    2. Beam-splitting Mirror (Dichroic Mirror): Reflects light below 510 nm and transmits light above 510 nm.
    3. Second Barrier Filter: Allows the specific green FITC emission to pass (520-560 nm).

Immunofluorescence Microscopy Techniques

  • Purpose: Used for detecting specific molecules inside fixed cells, reliant on available antibodies.

Types of Immunofluorescence Microscopy

  1. Direct Immunofluorescence Microscopy (suitable for high abundance proteins)

    • Procedure:
    1. Cells are fixed using glutaraldehyde or formaldehyde, preserving their structures by reacting with free -NH2 groups.
    2. Cells are permeabilized to allow antibody access.
    3. FITC-conjugated antibody is added, which binds to the target protein.
    4. Observe under the fluorescence microscope.
    5. Cells are incubated with 1% w/v BSA to minimize non-specific binding.

  2. Indirect Immunofluorescence (suitable for low abundance proteins)

    • Procedure:
    1. A primary antibody (1o Ab) binds to the target protein.
    2. A FITC-conjugated secondary antibody (2o Ab) binds to the primary antibody; multiple secondary antibodies can bind to one primary antibody for signal amplification.
    3. Cellular fixation and permeabilization are performed similar to direct immunofluorescence.
    4. Observe under the fluorescence microscope.

Visualisation Examples

  • Examples of visualizing proteins using antibodies:
    • Actin: detected using FITC-Anti-actin antibody.
    • Mitochondria: stained using Mitotracker.
    • DNA: stained with DAPI.

Co-localization Studies

  • Purpose: To visualize the localization of two different proteins simultaneously using different fluorescent markers.
  • Example Configuration:
    • DNA detected using a primary antibody and goat anti-rabbit secondary labeled with FITC.
    • Cytochrome c detected with a primary antibody and goat anti-mouse secondary labeled with TRITC.
    • Merging images shows co-localization of the proteins.

Epitope Tags

Definition and Use

  • Epitope Tags: Short peptide sequences (approximately 10-15 amino acids) genetically engineered into proteins to facilitate detection.
  • Application: Useful when no specific antibody is available to detect a target protein.
  • Commercial Availability: Antibodies available to bind specifically to these tags.
  • Importance: Small size ensures minimal impact on protein structure and function. Common applications include:
    • Immunoblotting (Western Blotting)
    • Immunoprecipitation
    • Immunofluorescence microscopy

Common Types of Epitope Tags

  1. Myc Tag (EQKLISEEDL): 10 amino acid segment from human myc proto-oncogene; detected with anti-Myc antibody.
  2. HA Tag (YPYDVPDYA): 9 amino acid segment from human influenza virus HA protein; detected with anti-HA antibody.
  3. His6 Tag (HHHHHH): Structure allows binding to metal ions; useful in affinity chromatography and detectable with anti-His antibody.

Vector Systems for Epitope-Tagged Proteins

  • Various commercial vectors are available for expressing epitope-tagged proteins effectively, showcasing specific sequences and components necessary for successful expression.

Transient Transfection

  • Definition: Introduction of exogenous DNA (like plasmids) into cells.
  • Utility: Analyzing gene function, regulation, and protein function.
  • Characteristics: Transiently transfected genes are expressed only temporarily and are not integrated into the host genome.

Cell Cycle Analysis by Flow Cytometry

  • Phases of Cell Cycle: Identified phases include M phase, G1 phase, S phase, and G2 phase, which can be analyzed quantitatively by flow cytometry to determine the relative amount of DNA in cells.

Bromodeoxyuridine (BrdU) Incorporation Assay

  • BrdU: A synthetic nucleoside used to measure DNA synthesis in actively dividing cells.
  • Application: Identifying S-phase cells, e.g., incorporation measured in HeLa cells, with BrdU positive indicating cells undergoing replication.

Summary of Key Concepts

  • Fluorescent Dyes: A wide variety available for staining macromolecules and organelles in both fixed and live cells.
  • Immunofluorescence Microscopy: Essential techniques for determining the intracellular localization of proteins in fixed cells through direct and indirect methods.
  • Epitope Tags: Important for scenarios where specific antibodies are lacking to study protein expression, localization, or binding interactions.