lecture 4 - microscopy Flashcards
why would the the same protein vary significantly in density after centrifugation?
it has an attached lipid or carbohydrate
what is SDS-Polyacrylamide Gel Electrophoresis used for?
separation of proteins
what is the function of sodium dodecyl sulfate?
an ionic detergent that denatures proteins by binding to hydrophobic side chains and destabilizing its conformation
in SDS-PAGE, proteins are separated according to…
size / mass
what is an antibody assay?
using tagged antibodies to recognize a protein antigen
why would a primary and secondary antibody be used in an antibody assay?
permits very high sensitivity in detection of protein of interest
why would an antibody be unable to bind to an antigen?
protein was unfolded, contained post-translational modifications, or epitope is bound to another molecule
what is western blotting?
used to separate proteins AND identify a protein of interest
to detect an antigen, western blotting uses…
a primary antibody and a secondary antibody
what is the resolution of a light microscope?
0.2 μm
2 methods for imaging live cells / unstained tissues
phase-contrast microscopy and differential-interference-contrast
phase-contrast microscopy
useful for observing single cell, thin cell layers, and location and movement of organelles in live cells
DIC microscopy
used for extremely small details, thick objects, and organelles
hematoxylin
dye that binds to basic amino acids and DNA/RNA and stains blue
eosin
dye that binds to acidic amino acids and stains pink
fluorescence microscopy
reveals locations and dynamics of fluorescent molecules
FRAP
stands for “fluorescence recovery after photobleaching”
1. use a high-intensity light to bleach the light-emitting molecules; no fluorescence and will look dark
2. if the molecules are in dynamic equilibrium with unbleached molecules, the bleached molecules will be replaced by unbleached ones and fluorescence will come back
FRET
stands for “Forster resonance energy transfer”
1. uses pair of fluorescent proteins in which the emission wavelength of the first is close to the excitation wavelength of the second
2. very sensitive to small changes in distance and is not detectable at distances greater than 10 nm
transmission electron microscopy
lenses focus a high-velocity electron beam onto sample and focus electrons that pass through the sample onto a viewing screen
can living material be imaged by electron microscopy?
no
how can you view cells and tissues in electron microscopy?
by cutting them into thin sections
how do you prepare thin sample for electron microscopy?
chemically fix, dehydrate, and impregnate with liquid that hardens, and cut into sections about 5-100 nm; stain with heavy metals to see structures
cryoelectron microscopy
transmission electron microscopy but with frozen sample; can view hydrated, unfixed, unstained samples; can generate a three-dimensional model
cryoelectron tomography
allows researchers to determine the three-dimensional architecture of organelles / whole cells
scanning electron microscopy
view surfaces of metal-coated specimens; three-dimensional; resolving power of around 10 nm
