Protein Denaturation

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Protein Denaturation

• denature loss of structure due to protein
  unfolding
• unfolding leads to loss of function

Folded
Unfolded
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Sodium Dodecyl Sulfate (SDS)

• strongly denaturing detergent
• disrupts 2o, 3o, and 4o structures
• binds and confers negative charge to protein
• charge is proportional to mass

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Polyacrylamide gel electrophoresis can occur
under both denaturing and reducing conditions
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Polyacrylamide Gel Electrophoresis (PAGE)
Molecules are separated by size in an electric
field Smaller molecules migrate more rapidly
through porous gel matrix
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Acrylamide Polymerization
Polyacrylamide is a useful polymer for gel
electrophoresis because both the concentration
and cross-linking of the gel can be controlled
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Proteins are detected in PAGE gels by staining or
autoradiography
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Isoelectric Focusing relies on the migration of
charged proteins in an electric field
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Immunoblotting

• aka Western Blotting
• use Ab to detect protein after electrophoresis
• 1. Protein electrophoresis
• 2. Transfer proteins to membrane
• - electrophoretically
• - MeOH to reduce SDS
• 3. Incubate membrane with antibody
• 4. Extensive washing
• 5. Detect bound antibody
• - radiolabeled 2nd Ab or protein A
• - ELISA

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Western blotting with antibodies to detect
specific proteins
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Antibodies

• Analytical Techniques
• Utilizing Antibodies
• flow cytometry
• gel electrophoresis
• immunoprecipitation (IP)
• immunoblotting
• microscopy
• immunofluorescence (IFA)
• electron microscopy
• ELISA

• antibodies bind proteins with high specificity
  and affinity
• affinity chromatography
• analytical techniques

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Antibodies can be generated against protein
epitopes
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Antibodies can bind proteins with very high
affinities
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Polyclonal and monoclonal antibodies
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The Enzyme-Lined Immunosorbent Assay (ELISA)
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Conventional ELISA

• bind protein to membrane or 96-well microplate
• neg. (and pos.) controls
• purity?
• incubate with 1o and 2o antibodies
• use soluble chromogenic substrates in 96-well
  plates
• quantify Ag or Ab

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• measure absorbance with ELISA microplate reader

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Screening Expression Libraries

• prepare library in expression vector
• cDNA better than gDNA
• 6X possible reading frames
• screen with protein based probe
• antibody/western blot
• ligand binding or other activity

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Indirect Immunocytochemistry
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Stain for DNA.
What is fluorescence?
Energy of light is inversely proportional to the
wavelength. For example, violet light has higher
energy than red light. Fluorescent molecules are
special molecules that absorb incident light of a
particular wavelength then emit light of a longer
wavelength. The absorption of light causes
electrons in the molecule to become highly
energetic. When the highly energetic electrons
return to their original state, they emit light.
Wave length of incident light must be shorter
than the wavelength of emitted light since some
energy is unavoidably lost as heat.
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Fluorescence Microscope
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Fluorescence image of a cell in mitosis labeled w
3 tags Green fluorescein-labeled anti-tubulin
(MTS) Red anti- centromere protein Blue
DAPI (DNA)
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Images of Conventional (A) vs Confocal (B)
Fluorescence (Drosophila embryo stained w
fluorescent probe against actin)
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Specific markers for specific cells