Molecular biology of the cell, 6th
edition
Chapter 8
In vivo (in the living organism) & in vitro (in glass)
Hybrid cell technology
Hybridomas
Monoclonal antibody
Preparative ultracentrifuge, which rotates extracts of broken cells at high speeds. This
separates cell components by size and density.
Velocity sedimentation & equilibrium sedimentation
Column chromatography – a mixture of proteins in solution is passed through a column
containing a porous solid matrix. Depending on the choice of matrix, proteins can be
separated according to charge (ion-exchange chromatography), their hydrophobicity
(hydrophobic chromatography), their size (gel-filtration chromatography), or their ability to
bind to particular small molecules or to other macro-molecules (affinity chromatography).
Most individual proteins represent less than 1/1000 of the total cell protein, it is usually
necessary to use several different types of column sin succession to attain sufficient purity.
With affinity chromatography being the most efficient.
Epitope tagging for the purification of proteins.
Sodium dodecyl sulfate (SDS) & beta-mercaptoethanol.
SDS polyacrylamide-gel electrophoresis (SDS-PAGE)
First dimension of two-dimensional polyacrylamide-gel electrophoresis (separation of protein
molecules by isoelectric focusing).
Two-dimensional polyacrylamide-gel electrophoresis
Western blotting, or immunoblotting (detecting proteins)
Mass spectrometer
Fluorescence anisotropy, a change in the polarized light that is emitted by a fluorescently
tagged protein in the bound and free states.
The synthesis of cDNA
PCR
PCR uses repeated rounds of strand separation, hybridization and synthesis to amplify DNA.
Chapter 9
Electron microscopy (0.1 nm to 2 mm)
Light microscope (12 nm to 2 mm)
Interference between light waves
How the limit of resolution is defined
Limit of resolution – depends on the wavelength and the numerical aperture of the lens
system used. The wider the microscope opens its eye, the more sharply it can see.
Numerical aperture
, Bright-field microscope, dark-field microscope, phase-contrast microscope & differential-
interphase-contrast microscope
In situ hybridization
Fluorescence and fluorescence microscope
Fluorescent probes
Spindle microtubules are revealed with a green fluorescent antibody, centromeres with a red,
and DNA of the condensed chromosomes with the blue.
DAPI
Fluorescent nanoparticles or quantum dots
Indirect immunocytochemistry
Confocal fluorescence microscope (produces clear optical sections and three-dimensional
data sets)
Multiphoton imaging (takes advantage of the two-photon effect, can obtain sharp images, till
depth of 250 micrometer within a specimen, valuable for living tissues studies)
Green fluorescent protein (GFP) (barrel like protein)
The brainbow mouse
Fluorescence resonance energy transfer (FRET)
Photoactivation is the light-induced activation of an inert molecule to an active state.
Fluorescence recovery after photobleaching (FRAP) a strong focused pulse of laser light will
extinguish, or bleach, the fluorescence of GFP.
The point spread function of a lens determines resolution.
Super resolution microscopy can be achieved by reducing the size of the point spread
function.
Single fluorescent molecules can be located with great accuracy.
Chapter 14
The chemiosmotic process occurs in two linked stages, both of which are performed by
protein complexes in a membrane.
Stage 1 – electron-transport chain & electrochemical gradient across membrane
Stage 2 – ATP synthase
Electron-transport processes (in mitochondria and chloroplasts)
Mitochondrion structure (outer membrane, crista space, intermembrane space, inner
boundary membrane, cristae, crista junction, matrix)
Energy conversion in the mitochondria (summary)
The major net energy conversion catalyzed by the mitochondrion.
Combustion of H2O -> nearly all of the energy would be released in the form of heat.
Biological oxidation -> about half of the released energy is stored in a form useful to the cell
by means of the electron-transport chain in the crista membrane of the mitochondrion.
Electrochemical proton gradient across the inner mitochondrial membrane (H+ gradient, pH
gradient)
How redox potentials are measured.
Quinone (Q) is a small hydrophobic molecule that is freely mobile in the lipid bilayer.
Quinone electron carriers - Ubiquinone picks up one H+ in two steps
edition
Chapter 8
In vivo (in the living organism) & in vitro (in glass)
Hybrid cell technology
Hybridomas
Monoclonal antibody
Preparative ultracentrifuge, which rotates extracts of broken cells at high speeds. This
separates cell components by size and density.
Velocity sedimentation & equilibrium sedimentation
Column chromatography – a mixture of proteins in solution is passed through a column
containing a porous solid matrix. Depending on the choice of matrix, proteins can be
separated according to charge (ion-exchange chromatography), their hydrophobicity
(hydrophobic chromatography), their size (gel-filtration chromatography), or their ability to
bind to particular small molecules or to other macro-molecules (affinity chromatography).
Most individual proteins represent less than 1/1000 of the total cell protein, it is usually
necessary to use several different types of column sin succession to attain sufficient purity.
With affinity chromatography being the most efficient.
Epitope tagging for the purification of proteins.
Sodium dodecyl sulfate (SDS) & beta-mercaptoethanol.
SDS polyacrylamide-gel electrophoresis (SDS-PAGE)
First dimension of two-dimensional polyacrylamide-gel electrophoresis (separation of protein
molecules by isoelectric focusing).
Two-dimensional polyacrylamide-gel electrophoresis
Western blotting, or immunoblotting (detecting proteins)
Mass spectrometer
Fluorescence anisotropy, a change in the polarized light that is emitted by a fluorescently
tagged protein in the bound and free states.
The synthesis of cDNA
PCR
PCR uses repeated rounds of strand separation, hybridization and synthesis to amplify DNA.
Chapter 9
Electron microscopy (0.1 nm to 2 mm)
Light microscope (12 nm to 2 mm)
Interference between light waves
How the limit of resolution is defined
Limit of resolution – depends on the wavelength and the numerical aperture of the lens
system used. The wider the microscope opens its eye, the more sharply it can see.
Numerical aperture
, Bright-field microscope, dark-field microscope, phase-contrast microscope & differential-
interphase-contrast microscope
In situ hybridization
Fluorescence and fluorescence microscope
Fluorescent probes
Spindle microtubules are revealed with a green fluorescent antibody, centromeres with a red,
and DNA of the condensed chromosomes with the blue.
DAPI
Fluorescent nanoparticles or quantum dots
Indirect immunocytochemistry
Confocal fluorescence microscope (produces clear optical sections and three-dimensional
data sets)
Multiphoton imaging (takes advantage of the two-photon effect, can obtain sharp images, till
depth of 250 micrometer within a specimen, valuable for living tissues studies)
Green fluorescent protein (GFP) (barrel like protein)
The brainbow mouse
Fluorescence resonance energy transfer (FRET)
Photoactivation is the light-induced activation of an inert molecule to an active state.
Fluorescence recovery after photobleaching (FRAP) a strong focused pulse of laser light will
extinguish, or bleach, the fluorescence of GFP.
The point spread function of a lens determines resolution.
Super resolution microscopy can be achieved by reducing the size of the point spread
function.
Single fluorescent molecules can be located with great accuracy.
Chapter 14
The chemiosmotic process occurs in two linked stages, both of which are performed by
protein complexes in a membrane.
Stage 1 – electron-transport chain & electrochemical gradient across membrane
Stage 2 – ATP synthase
Electron-transport processes (in mitochondria and chloroplasts)
Mitochondrion structure (outer membrane, crista space, intermembrane space, inner
boundary membrane, cristae, crista junction, matrix)
Energy conversion in the mitochondria (summary)
The major net energy conversion catalyzed by the mitochondrion.
Combustion of H2O -> nearly all of the energy would be released in the form of heat.
Biological oxidation -> about half of the released energy is stored in a form useful to the cell
by means of the electron-transport chain in the crista membrane of the mitochondrion.
Electrochemical proton gradient across the inner mitochondrial membrane (H+ gradient, pH
gradient)
How redox potentials are measured.
Quinone (Q) is a small hydrophobic molecule that is freely mobile in the lipid bilayer.
Quinone electron carriers - Ubiquinone picks up one H+ in two steps
