lecture 3 biochemistry (week 1) Flashcards
(27 cards)
describe the antibody structure
4 chains; 2 heavy (H) chains (blue)
and 2 light (L) chains (red), linked by disulfide bonds.
the H and L chains come together to form Fab domains, which have the
antigen-binding sites at the ends. The two H chains form the Fc domain.
notice that the Fab domains are linked to the Fc domain by flexible linkers
what types of antibodies can be used as tools?
both monoclonal and polyclonal antibodies
describe polyclonal antibodies
heterogeneous
mixtures of antibodies, each specific for
one of the various epitopes on an antigen
describe monoclonal antibodies
are all identical,
produced by clones of a single
antibody-producing cell.
They recognize one specific epitope.
describe the preparation of monoclonal antibodies
hybridoma cells are formed by the
fusion of antibody-producing cells
and myeloma cells. The hybrid cells
are allowed to proliferate by
growing them in selective medium.
then screened to
determine which ones produce
antibody of the desired specificity
how can proteins be detected and quantified using an enzyme-linked immunosorbent assay?
antibodies can be used as exquisitely specific analytic reagents to quantify the amount of a protein or other antigen present in a biological sample. The enzyme-linked immunosorbent assay (ELISA) makes use of an enzyme that reacts with a colourless substrate to produce a coloured product.
how are antibodies used in sds page immunoblot?
electrophoretic separations are nearly always carried out in porous gels (or on solid supports such as paper) because the gel serves as a molecular sieve that enhances separation
gels can be stained - silver nitrate or a dye such as Coomassie blue
what does western blotting permit in sds page immunoblot?
detections of proteins separated by gel electrophoresis
what is the order of the objects viewable on a microscope?
atom, small molecule, globular protein, virus, ribosome, bacterium, animal cell, plant cell, frog egg
what is the light path in a microscope?
eye –> eyepiece –> tube lens –> objective –> specimen –> condenser –> iris diaphragm –> light source
how does bright field microscopy work?
light is transmitted straight through the specimen
how does phase contrast microscopy work?
phase alterations of light transmitted through the specimen are translated into brightness changes
how does differential interference contrast microscopy?
which highlights edges where there is a steep change of refractive index
how does dark field microscopy work?
the specimen is lit from the side and only the scattered light is seen
how do specific probes help understand biology?
the possibility to visualize the distribution of different RNAs in tissues using in situ hybridization
what are the expression patterns in probes>
wingless - yellow
engrailed - blue
short gastrulation - red
intermediate neuroblasts defective - green
muscle specific homeobox - purple
how does direct immunocytochemistry work?
detection method is very sensitive - many molecules of the secondary antibody
recognise each primary antibody.
the secondary antibody is covalently coupled to a marker
molecule that makes it readily detectable
what are the most commonly used marker molecules?
fluorescent probes (for fluorescence microscopy)
enzyme horseradish peroxidase (for either conventional light microscopy or electron
microscopy)
colloidal gold spheres (for electron microscopy)
the enzymes alkaline
phosphatase or peroxidase (for biochemical detection
what are actin filaments?
fluorescence micrograph of a cell shows actin filaments stained green using an antibody specific to actin
what is the theory of flurochromes?
an orbital electron of a fluorochrome molecule can be raised to an excited state following
the absorption of a photon.
fluorescence occurs when the electron returns to its ground state
and emits a photon of light at a longer wavelength.
too much exposure to light, or too bright a
light, can also destroy the fluorochrome molecule, in a process called photobleaching
describe the excitation and emission of different fluorochromes
maximum excitation and emission wavelengths of several fluorescent probes
the photon emitted by a fluorescent molecule is necessarily of lower energy (longer wavelength) than the absorbed photon and this accounts for the difference between the excitation and emission peaks
DAPI is widely used as a general fluorescent DNA probe, which absorbs ultraviolet light and fluoresces bright blue
FITC is an abbreviation for fluorescein isothiocyanate, a widely used derivative of fluorescein, which fluoresces bright green
the other probes are all commonly used to
fluorescently label antibodies and other proteins
what is the fluorescence microscope structure?
a filter set consists of two barrier filters and and a dichroic
(beam-splitting) mirror
describe microtubules, centromeres, and chromosomes
composite micrograph of a cell in mitosis
three different fluorescent probes:
spindle microtubules green fluorescent
antibody,
centromeres with a red fluorescent
antibody
DNA of the condensed chromosomes
with the blue fluorescent dye DAPI.
