How Cells Are Studied

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Chapter 4

• How Cells Are Studied

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Studying Cells

• Various ways to study cells, techniques used in
  vitro and in vivo
• Must grow enough cells to accomplish the goal of
  the study, same goes for tissue

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Cells In Culture

• Developed so that the biochemical processes can
  be unraveled
• Usually needs to be from one cell type
• Methods have been developed to separate cells and
  parts of cells

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Cells Grown in Culture

• In vitro - cells in culture
• In vivo - cells in an organism
• Explants culturing small pieces of tissue
• Primary culture cells directly from a tissue
• Secondary culture cells removed from a primary
  culture called passing of cells
• Cells are grown in serum free, chemically defined
  medium, so you can add back things to study the
  effects on cells growth factors

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Additional Cell Culture Terms

• Cell Line a culture that has undergone a
  genetic change and have become immortal
• Transformed Cell Line normal cells that have
  been infected with a tumor virus or a chemical so
  that they become more like cancer cells
• Usually free floating, unattached
• Higher density than other cells
• Cell Cloning allow one cell to divide by itself
  to fill the whole dish all the cells are
  identical

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Disruption of Cells

• Usually use fetal or neonatal tissues
• Add proteases and a chelator to break down the
  extracellular matrix and release the cells
• Agitation to help break up cells
• Goal get a population of cells for analysis
  that can be perpetuated

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Separation Methods

• Affinity surfaces are helpful
• Antibodies can be used to find one cell type
• Cells can be attached to collagen coated plate
• Use polysaccharide beads to bind to cells
• Wash all the non-attached cells away and have a
  pure culture
• Fluorescence activated cell sorter
• Machine that uses a fluorescently labeled Ab to
  separate fluorescent cells from unlabeled cells
• Also can determine the amount of fluorescence on
  each cell

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Fluorescence Activated Cell Sorter (FACS)
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Hybrid Cells
Used to map human chromosomes
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Cell Fractionation Terms

• Homogenate or extract suspension of broken
  cells
• Hopefully biochemical properties intact
• Preparative ultracentrifuge separates cell
  components by high speed based on size and
  density
• Large parts move faster or sediment
• Usually requires extended time
• Cell-free system fractionated cell homogenates
  that maintain biological function
• Used to determine many things including protein
  synthesis and virus capsid formation

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Ultracentrifuge
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Centrifugation

• Three types of centrifugation
• Differential
• Equilibrium gradient
• Density gradient
• Sample prep bust up the cells in a cold
  isotonic solution to end up with a homogenate
  all the cellular components in a mixture

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Differential Centrifugation

• Separation is based on size and/or density (pp
  327-328)
• Sedimentation Coefficient measurement of how
  rapidly the particle sediments when centrifuged

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Using Differential Centrifugation
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Density Gradient Centrifugation

• Also called rate-zonal centrifugation (328-329)
• Place sample on a gradient such as sucrose (small
  concentration range)
• After centrifugation (low speeds), bands form
  where the organelle has the same density as the
  sucrose solution around it
• Larger fragments move farther than the smaller
  ones

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Equilibrium Density Centrifugation

• Also called buoyant density centrifugation
  (329-330)
• Place sample on a gradient such as sucrose (large
  concentration range)
• After centrifugation (high speeds), bands form
  where the organelle has the same density as the
  sucrose solution around it
• Denser fragments move farther than the others
• Usually done after one of the other types of
  centrifugation steps

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Chromatography

• Partition chromatography
• Spot the sample on paper, use a solvent mixture
  to separate the molecules, based on relative
  solubility in the solvent
• Column chromatography
• Column filled with porous matrix (stationary
  phase) and separated based on the interaction of
  with sample
• Move through with liquid (mobile phase)
• High performance liquid chromatography
• Use a fine matrix and high pressure to move the
  mobile phase through the column

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Partition Chromatography
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Column Chromatography
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4 Types of Chromatography
Hydrophobic chromatography
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Protein Electrophoresis

• Proteins usually have positive or negative net
  charge based on their aa side chains
• Adding SDS and mercaptoethanol to sample
• Add electrical charge to separate them
• Stain the proteins to visualize
• Can also determine the size of proteins

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Combining Chromatography and SDS-PAGE
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Two-Dimensional Gel Electrophoresis

• Has 2 separation procedures
• Based on isoelectric focusing the pH at which
  the protein has no net charge in an electric
  field
• Almost all proteins move to a unique site

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2 Separation Steps

• 1st phase use a small tube of acrylamide and
  sample in mercaptoethanol and urea (charge
  unchanged)
• 2nd phase SDS-PAGE

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Western Analysis

• Technique used to identify a particular protein
• Transfer the proteins in the SDS-PAGE onto a
  nitrocellulose membrane
• Block unoccupied sites with albumin and milk
• Probe with an antibody to your particular
  protein, usually with a detection molecule

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2D Gel and Western
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Peptide Mapping

• Fragment the protein
• Separate the fragments
• Isolate and sequence the AA residues
• Allow us to determine the sequence of the protein

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Peptide Cleaving Reagents
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Modern Way to Sequence Proteins

• Isolate the protein
• Sequence the first 10-20 amino acids by automated
  sequencer
• Create a DNA probe from AA sequence data
• Isolate the gene using the DNA probe
• Sequence the gene
• Use software to determine the AA sequence

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Radioactive Molecules as Probes

• Isotope have extra neutrons in the nucleus and
  therefore the atom is unstable, giving off
  radiation
• Detect the radiation emitted by Geiger counter,
  scintillation counter or autoradiography
• Use P, I, S, C, Ca and H as isotopes

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Autoradiography (429)

• Cells are exposed to an isotope
• The sample is processed for light or electron
  microscopy
• Subject the slide to a photographic emulsion
• Allow the isotope to degrade over time
• Develop the emulsion like a negative
• Look for the deposition of silver grains on the
  slide for location of where the isotope is located

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Pulse-Chase Experiments
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Pulse-Chase Experiment

• Dark spots, location of isotope, is first seen in
  the Golgi apparatus
• 45 minutes later it is seen in the secretory
  vesicles

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Antibodies as Labels

• Antibodies can be generated to specific proteins
  (antigen) and then used to locate that protein
• If abundant then do a direct label
• If not, do an amplification step to increase the
  visibility

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Making of Monoclonal Ab