Methods for studying proteins

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Methods for studying proteins
Protein purification Centrifugation Differentia
l, Rate-zonal Column Chromatography Ion
exchange, Gel filtration, Affinity Protein
detection Enzyme assay (functional), SDS-PAGE,
Coomassie and silver stains, Antibodies Prepar
ation, Westerns, Immunoprecipitation Labeling
with radioactive compounds Pulse-chases
analysis Protein analysis Edman degradation,
Mass Spectrometry X-ray crystallography NMR Cry
oelectron Microscopy
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Protein detection Labeling with radioactive
compounds
Commonly used radioisotopes Tritium 3H Carbon-14
14C Phosphorus-32 32P Sulfur-35 35S Iodine-125
125I
Table 3-2
Purified proteins can be chemically iodinated
with 125I to make them radioactive. Radioactive
proteins can be purified from cells metabolically
labeled with radioactive amino acids (14C amino
acids or 35S methionine and cysteine).
Radioactivity is incorporated during protein
synthesis. Cells metabolically labeled with
32P will incorporate this radioactive compound
into DNA, RNA, phospholipids and phosphoproteins
(posttranslational addition of 32P by a
kinase) Radioactive molecules can be detected
quantitatively using a scintillation counter, or
qualitatively by exposure to X-ray film.
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Protein detection Labeling with radioactive
compounds. Pulse-chases analysis
A. In this experiment, yeast cells were
pulse-labeled with 35S-methionine for 5 minutes
and then chased will a 100 fold excess of cold
methionine. At the chase times indicated, an
aliquot of cells were lysed and the vacuolar
(lysosomal) protein carboxypeptidase Y (CPY) was
immunoprecipitated. CPY forms were resolved by
SDS-PAGE and exposed to x-ray film. B. CPY is
initially synthesized in the ER as a high
molecular weight precursor containing a cleavable
signal sequence (not shown) and four N-linked
oligosaccharides (vertical lines). The p1 form is
transported to the Golgi where the N-linked
oligosaccharides are further modified (circles)
to produce p2 CPY. Upon arrival in the vacuole,
the propiece (black) is removed by vacuolar
proteases (PEP4) to generate the mature,
enzymatically active CPY (mCPY).
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Microscopy
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Microscopy
Light Microscopy Resolution, Contrast Brightfiel
d, Phase-Contrast, DIC, Fluorescence
Microscopy Laser Scanning Confocal
Microscopy Electron Microscopy
(EM) Transmission EM Scanning EM
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The ability of light and electron microscopes to
resolve particles of different sizes
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Resolution The minimum distance between two
distinguishable objects (D)
The optical pathway in a compound microscope
10x
100x
Ocular x objective gives final magnification
(1000x)
D 0.61 l / N sin a l the wavelength of
light N refractive index of air (1) or oil
(1.5) a angular aperture (angle of light that
can be collected by the objective lens) N sin a
the numerical aperture (NA) of the lens A good
oil immersion objective lens has an NA of 1.4
5-2
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Resolution The minimum distance between two
distinguishable objects (D) Contrast allows
objects to be distinguishable. Contrast
Contrast Contrast Contrast Contrast
Contrast Cells and subcellular particles usually
have very little contrast making them hard to see
using a microscope with standard brightfield
optics. Contrast can be improved through use
of 1) Optical tricks Phase-contrast,
Differential Interference Contrast (DIC or
Nomarski) 2) Stains Chemical (hematoxylin,
eosin) Cytochemical (colored products of an
enzyme reaction that precipitate) Fluorescent
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1) Optical tricks Phase-contrast, Differential
Interference Contrast (DIC)
Interference
Amplification
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Phase-contrast optics
5-12
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(b) Nomarski optics or Differential Interference
Contrast (DIC) Separate an combine light waves
to produce interference patterns Light is
polarized before it passes through a specimen and
only the light rays that were rotated by the
specimen are allowed to form the image plane.
5-13
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Dealing with thick samples (tissues)
Tissue sectioning
Fixative
Microtome
cross-link proteins together
5-3
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Staining cells Improves contrast Nonspecific
stains such as hematoxylin or eosin Specific
stains for molecule or organelle localization
Cytochemical or histochemical stain for an
enzymes activity (succinate dehydrogenase in the
mitochondria)
5-4
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Fluorescence microscopy Protein localization
5-6
Skin fibroblast stained with a primary actin
antibody and a secondary antibody coupled to a
green fluorescent molecule
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Commonly used fluorescent molecules coupled to
different probes
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Fluorescent molecules have unique excitation
(absorption) and emission spectra. Filters can
be applied to a microscope that will only allow
light of a specific range of wavelengths to pass
to the eyepiece.
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Epi-fluorescence microscope
5-5
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Laser Scanning Confocal Fluorescence Microscopy
Standard fluorescence microscopy
Confocal fluorescence microscopy
Microtubules in a sea urchin egg
5-9
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Standard fluorescence microscopy
Confocal fluorescence microscopy
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Z-axis scan of a pollen granule using confocal
microscope
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Transmission Electron Micrograph Fluorescence
Micrograph
5-1
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Transmission Electron Microscope
A strong vacuum is maintained in the chamber
5-15