Laboratory methods

Phenomics: The Next Frontier

• Phenomics integrates genomics and environmental influence to predict an organism's physiological outcome.
• The "physiome" refers to the complete set of physiological processes within a living organism.

Omics: A Scalar Approach to Life

• Omics approaches involve investigating the complete sets of different types of molecules and biological structures.
• These range from genomics (DNA) to transcriptomics (RNA) and proteomics (proteins) to metabolomics (metabolites) and cellomics (cells).
• Phenomics sits at the top, integrating all these levels to study the complete physiological function.
• This approach aids in understanding biological complexities and offers a pathway for personalized medicine.

Omics in Drug Discovery

• Phenomics and physiomics provide an alternative approach to traditional drug discovery by studying entire physiological systems.
• This approach utilizes bioassays within an intact physiological milieu, avoiding the need for prior knowledge of molecular targets.
• Studying phenotypes and physiomies gives insight into drug effect on complete biological systems, enhancing drug efficacy and precision.

Microscopy: Seeing cells

• Microscopy is a fundamental tool in cell biology, offering diverse techniques to see and analyze living cells.
• The optical microscope, using visible light and lenses, was the first, while advancements have led to various high resolution imaging methods.

Types of Microscopy

• Different light microscopy techniques exploit various properties of light to visualize cells, including fluorescence and confocal microscopy.
• Electron microscopy offers even higher resolution via electron beams, enabling the visualization of intracellular structures and even molecules.

Cell Preparation for Microscopy

• Cells often require preparation before microscopy, which can include:
  • Fixation: Stabilizing cells to preserve structure.
  • Staining: Employing dyes to highlight specific cellular components.
  • Sectioning: Thinly slicing cells for viewing internal structures.

Immunofluorescence: Staining with Antibodies

• Immunofluorescence uses fluorescent probes combined with antibodies to locate specific molecules within cells.
• Antibodies, known as immunoglobulins, are produced by immune cells, targeting specific structures called antigens.
• This technique allows researchers to visualize and analyze the location and abundance of specific molecules within cells.

Antibodies: Defense Proteins

• Antibodies are produced by white blood cells called B lymphocytes in response to the presence of antigens.
• They bind to and neutralize pathogens (bacteria, viruses) or mark them for destruction by immune cells.
• Antibodies are Y-shaped proteins with a constant region (C), common to the antibody type, and variable regions (V) that bind distinct antigens.

Antigens: Targets of Antibodies

• Antigens are molecular structures that are specifically recognized and bound by antibodies.
• Each antibody binds to a specific epitope, a part of the antigen, allowing the immune system to identify and respond to specific threats.
• Antibodies can be engineered to target specific antigens for scientific and medical purposes.

Monoclonal Antibodies: Mass-Produced Specificity

• Monoclonal antibodies (mAbs) are laboratory-produced antibodies that bind to the same epitope, enabling high specificity and reproducibility.
• They are generated by cloning a single immune cell that produces a specific antibody, allowing for mass production for research and therapeutic applications.

Immunofluorescence: Direct vs. Indirect

• Direct Immunofluorescence: Fluorescent dyes are directly attached to primary antibodies that specifically bind to the target molecule.
• Indirect Immunofluorescence: Unlabeled primary antibodies bind to the target, followed by labeled secondary antibodies that bind to the primary antibodies, enhancing signal.

Fluorescent Microscopy: Imaging Specific Signals

• Fluorescent microscopy uses filters to detect specific wavelengths of light emitted by fluorescent probes, allowing distinct signals to be visualized.
• Different combinations of probes and filters enable the simultaneous imaging of multiple cellular structures and molecules.

Two-Dimensional Gel Electrophoresis: Separating Proteins

• 2D SDS-PAGE combines two techniques to separate proteins based on both size and charge:
  • Isoelectric focusing: Separating proteins based on their isoelectric point (the pH where the net charge is zero).
  • SDS-PAGE: Separating proteins based on molecular size using sodium dodecyl sulfate (SDS) and gel electrophoresis.

Western Blotting: Detecting Specific Proteins

• Western blotting identifies specific proteins within a complex mixture:
  • Proteins are first separated by size using SDS-PAGE.
  • Separated proteins are transferred to a membrane (nitrocellulose or nylon) using an electric current.
  • Specific proteins are then detected using labeled antibodies, which bind to their corresponding antigens.

Studying Enzymes:

• Enzyme kinetics studies the rate of enzyme-catalyzed reactions, which is influenced by enzyme, substrate, and environmental factors.
• Spectrophotometers measure the absorbance of light by solutions, allowing the progress of reactions to be monitored by changes in reactant or product concentration.
• The dependence of reaction velocity (v) on substrate concentration ([S]) is often described by a hyperbolic curve.
• Saturation: At high substrate concentrations, enzyme molecules are saturated, leading to a plateau in reaction velocity.
• Double-Reciprocal Plot (Lineweaver-Burk): A linear plot of 1/v vs. 1/[S] for easier determination of kinetic parameters like Vmax and Km.

DNA Recombinant Technology

• Restriction nucleases (enzymes) cleave DNA molecules at specific sequences, allowing for manipulation and analysis of DNA.
• This leads to the generation of restriction fragments, DNA pieces with specific lengths.

DNA Electrophoresis: Separating DNA Fragments

• DNA electrophoresis separates DNA fragments based on size using a gel matrix and an electric field.
• The smaller the fragment, the faster it migrates through the gel.

Southern Blotting: Identifying Specific DNA Sequences

• Southern blotting combines the processes of DNA digestion, electrophoresis, and hybridization to identify a specific DNA sequence within a complex mixture.
  • DNA is digested with restriction enzymes to create fragments.
  • Fragments are separated by size using electrophoresis.
  • DNA transferred from the gel to a membrane.
  • A labeled probe (single-stranded DNA) complementary to the target sequence hybridizes to the membrane.
  • Hybridization can be detected by autoradiography or chemiluminescence.