Lecture 3-4 Cell Biology Methodology

• A whole cell lysate is a single cell suspension of desired cells that is ready to be opened, producing a cell lysate/homogenate/extract.
• Cell lysate contains large and small molecules from the cytosol.
• Cell fractionation separates cellular components from one another and isolates organelles, usually done with a centrifuge.
• SDS-PAGE separates proteins in a mix by size, using gel electrophoresis.
• Coomassie blue uses a stain to detect all proteins in a gel, the stain binds to basic amino acids.
• The western blot technique uses antibodies to detect specific protein on a membrane.
• Coomassie blue is nonspecific, binds to basic amino acids, and can identify protein that has been purified.
• Western blots can show different size bands, larger bands indicate more signal which indicates more protein in the sample.
• Antibodies are proteins composed of 4 polypeptide chains, two identical light and two identical heavy chains.
• Antibodies have variable regions that can bind to an antigen.
• An antigen is a substance that stimulates an immune response.
• Antibodies are naturally made by immune systems.
• Transmission electron microscope (TEM) passes electrons through a specimen to create an image, showing internal structures in detail.
• Fluorescent tags can be added to DNA sequence to create fluorescent protein (fusion protein).
• A confocal microscope produces sharp, high-contrast images by eliminating out-of-focus light, often used for 3D imaging and studying dynamic processes in living cells.
• A light microscope generally shows lower resolution and lacks depth compared to electron microscopes, typically used for observing larger structures like cells and tissues.
• Scientists use SEM when they want detailed surface images, such as for studying the topography of materials or biological samples.
• Scanning electron microscope (SEM) scans a specimen's surface with electrons to create a 3D image, revealing surface features.
• Green fluorescent protein (GFP) helps detect proteins.
• Low energy gives us higher wavelength.
• A fluorescence microscope shows fluorescently labeled structures, used to study protein localization.
• Scientists typically use a TEM when they need high-resolution images of internal structures, like cell organelles or nanomaterials.
• High energy gives us a lower wavelength.
• Immunofluorescence uses antibody with fluorescent dye to detect specific protein.
• A major disadvantage of electron microscopy compared to light and fluorescence microscopy is that it is expensive and does not allow for live imaging.
• A scanning electron microscope (SEM) produces detailed 3D-like images of a sample's surface by scanning it with a focused electron beam, useful for observing surface topography and morphology.
• A confocal microscope builds 3D images with a laser beam.
• Emission of photon at longer wavelength.
• A fluorescent label in fluorescence microscopy has excitation and emission wavelengths.
• A transmission electron microscope (TEM) offers high-resolution images of internal structures by passing electrons through a thin specimen, used for detailed examination of ultrastructural features.
• Electron microscopes use a beam of electrons as their source, while light/fluorescence microscopes use light as their source.
• GST protein is attached to gene sequence to make fusion protein.
• A GST tag is used to purify a specific protein from a cell lysate because GST has a high affinity for GSH.
• Immunoprecipitation uses antibodies to purify protein complexes and asses protein-protein interactions.
• In an immunoprecipitation, a cell lysate is generated, cross-linked to stabilize protein-protein interactions, an antibody conjugated with bead is added, a centrifuge is used to pull down bead, and proteins not bound by antibody are discarded and proteins bound by antibody are purified.
• Monoclonal antibodies are ideal for drug treatment and scientific research because they have low variability between batches.
• Advantages of fluorescence microscopes include allowing visualization of specific structures, live cell imaging, and 3D, but disadvantages include being more expensive and fluorescence will fade over time.
• Myeloma cells are used in the process of making monoclonal antibodies because these hybrid cells produce antibodies that never stop dividing.
• Monoclonal antibodies have one antibody attached to antigen.
• Advantages of light microscopes include being cheap and being able to look at live cells, but disadvantages include specimen must be transparent and limit to magnification because light source.