lecture 4 - microscopy

Question 1

why would the the same protein vary significantly in density after centrifugation?

Answer 1

it has an attached lipid or carbohydrate

Question 2

what is SDS-Polyacrylamide Gel Electrophoresis used for?

Answer 2

separation of proteins

Question 3

what is the function of sodium dodecyl sulfate?

Answer 3

an ionic detergent that denatures proteins by binding to hydrophobic side chains and destabilizing its conformation

Question 4

in SDS-PAGE, proteins are separated according to…

Answer 4

size / mass

Question 5

what is an antibody assay?

Answer 5

using tagged antibodies to recognize a protein antigen

Question 6

why would a primary and secondary antibody be used in an antibody assay?

Answer 6

permits very high sensitivity in detection of protein of interest

Question 7

why would an antibody be unable to bind to an antigen?

Answer 7

protein was unfolded, contained post-translational modifications, or epitope is bound to another molecule

Question 8

what is western blotting?

Answer 8

used to separate proteins AND identify a protein of interest

Question 9

to detect an antigen, western blotting uses…

Answer 9

a primary antibody and a secondary antibody

Question 10

what is the resolution of a light microscope?

Answer 10

0.2 μm

Question 11

2 methods for imaging live cells / unstained tissues

Answer 11

phase-contrast microscopy and differential-interference-contrast

Question 12

phase-contrast microscopy

Answer 12

useful for observing single cell, thin cell layers, and location and movement of organelles in live cells

Question 13

DIC microscopy

Answer 13

used for extremely small details, thick objects, and organelles

Question 14

hematoxylin

Answer 14

dye that binds to basic amino acids and DNA/RNA and stains blue

Question 15

eosin

Answer 15

dye that binds to acidic amino acids and stains pink

Question 16

fluorescence microscopy

Answer 16

reveals locations and dynamics of fluorescent molecules

Question 17

FRAP

Answer 17

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

Question 18

FRET

Answer 18

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

Question 19

transmission electron microscopy

Answer 19

lenses focus a high-velocity electron beam onto sample and focus electrons that pass through the sample onto a viewing screen

Question 20

can living material be imaged by electron microscopy?

Answer 20

no

Question 21

how can you view cells and tissues in electron microscopy?

Answer 21

by cutting them into thin sections

Question 22

how do you prepare thin sample for electron microscopy?

Answer 22

chemically fix, dehydrate, and impregnate with liquid that hardens, and cut into sections about 5-100 nm; stain with heavy metals to see structures

Question 23

cryoelectron microscopy

Answer 23

transmission electron microscopy but with frozen sample; can view hydrated, unfixed, unstained samples; can generate a three-dimensional model

Question 24

cryoelectron tomography

Answer 24

allows researchers to determine the three-dimensional architecture of organelles / whole cells

Question 25

scanning electron microscopy

Answer 25

view surfaces of metal-coated specimens; three-dimensional; resolving power of around 10 nm