Key points

1
Cell Biology Techniques
Key points stages in analysis of cellular
process microscopy of fixed or live
cells approaches to identify proteins protein
purification techniques
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Mitosis in animal cells how does it work?
3
Steps in cell biological analysis of a
process 1. Describe it 2. Identify players 3.
Reconstitute in vitro 4. Test models in vivo
and in vitro
4
Microscopy excellent descriptive tool, 2 major
types

• Magnification, Contrast, Resolution
• Light Microscopy (200 nm)
• Brightfield
• Fluorescent
• Advanced
• Electron Microscopy (1 nm)
• Transmission
• Scanning

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5.1 The light microscope
Figure 5-2
Min. distance between objects (D)
0.61(wavelength) / (refractive index)(sin a)
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5.1 Brightfield microscopy

• Problem Most cells are colorless transparent
• To visualize structures ? stain with dyes
• Must preserve (fix), embed, section
• New problem ? these actions
• Alter cell structure/molecules
• Only give snapshot of dead cells

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5.1 Fluorescent microscopy

• Permits localization of specific cellular
  molecules
• Fluorescent dyes glow against dark background
• Dye may be indirectly or directly associated with
  
• the cellular molecule
• Multiple fluorescent dyes may be used
  simultaneously
• Cells may be fixed or living

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How to localize a protein with fluorescence
microscopy
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5.1 The fluorescent microscope
Figure 5-6
Figure 5-5
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5.1 Light microscopy of three-dimensional objects

• Confocal Scanning or Deconvolution Microscopy
• Generates 3D images of living cells
• Removes out-of-focus images ? optical sectioning
• Can look inside thick specimens (eggs, embryos,
  tissues)

Figure 5-9
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5.1 Advanced light microscopy

• Permits observation of transparent living cells
• Light phase shifts induced by specimen are used
  to
• generate contrast
• Phase contrast (refracted and unrefracted light)
• Differential interference contrast (two light
  beams)

Figure 5-14
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5.1 The transmission electron microscope
Figure 5-16
Figure 5-15
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Immuno-EM can localize a specific type of protein
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• Techniques to identify the proteins involved
• Pharmacological Approach find drugs that perturb
  process.
• Genetic Approach setup a selection or screen
  which allows identification of mutations that
  perturb the process
• Biochemical Approach reconstitute the process in
  a cell free system ("in vitro"), purify the
  molecules that are required for the in vitro
  assay to work

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• Pharmacological Approach find drugs that perturb
  process.
• specificity of inhibitor is important
• Particularly useful if the drug has a known
  specific mechanism of action (eg inhibition of a
  certain protein gt that protein is required for
  the process).
• drug may block by binding protein involved in the
  process, so even if mechanism of action is
  unknown the drug might be used to "fish out" the
  protein it binds (e.g. by affinity
  chromatography)
• affinity chromatography link drug (or any
  ligand) to beads in a column, pass cell lysate
  over column, proteins that bind column can then
  be eluted and analyzed (for example by SDS-PAGE)

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3.5 Separation of proteins by specific binding to
another molecule affinity chromatography
Figure 3-43c
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• Genetic Approach setup a selection or screen
  which allows identification of mutations that
  perturb the process
• specificity of selection or screen is important
• if mutation is known then the corresponding
  protein is implicated
• if mutation is not known the mutated gene may
  identified by complementation and sequence
  analysis

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• Biochemical Approach reconstitute the process in
  a cell free system ("in vitro"), purify the
  molecules that are required for the in vitro
  assay to work
• fidelity of the reconstitution is important (ie
  does in vitro assay in vivo?)
• to reconstitute a process the cells are
  permeabilized or fractionated
• subcellular fractionation typically involves
  homogenization, differential centrifugation, or
  gradient centrifugation
• centrifugation separates on the basis of mass and
  density
• to purify the proteins you need separation
  techniques and an assay

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• How are proteins purified?
• electrophoresis separates on the basis of
  charge/mass ratio. SDS-PAGE is very effective but
  involved denaturation of the protein sample. Two
  dimensional electrophoresis has even more
  resolving power.
• gel filtration beads with pores of defined
  sizes, separates on basis of mass ( shape)
  because molecules partition into pore as they
  pass through column
• ion exchange beads with defined charge,
  separates on basis of charge, elute w/ salt or pH
• affinity as above binding is to ligand on
  column. Note that if antibody is used it is also
  called immunoprecipitation.
• hydrophobic beads with acyl chains, separates on
  basis of hydrophobicity, elute with hydrophobic
  buffer

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3.5 Electrophoresis separates molecules according
to their chargemass ratio
SDS-polyacrylamide gel electrophoresis
Figure 3-41
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3.5 Separation of proteins by size gel
filtration chromatography
Figure 3-43a
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3.5 Separation of proteins by charge ion
exchange chromatography
Figure 3-43b