L2: Tools and techniques (cell imaging)

Question 1

what resolution can a normal transmitted light microscope give

Answer 1

0.2micrometres

Question 2

advantage of transmitted and fluorescence light microscopy

Answer 2

can both use live cells

Question 3

how can fluorecence microscopy help visualise specific cellular cojmponents

Answer 3

some dyes can bind specifically to organelles or other components

e.g. DAPI binds to DNA

Question 4

disadvantage of coupling fluorescence dyes with antibodies for imaging

Answer 4

cells have to be dead

Question 5

what is GFP

Answer 5

green fluorescent protein
can attach to specific proteins for fluorescence microscopy
can make it attach to proteins using molecular biology to add GFP coding sequence
to produce fusion protein

Question 6

advantage of GFP

Answer 6

visible in living cells

Question 7

potential problem of GFP

Answer 7

cuz it has many AAs cuz it’s a protein duh
it could alter the function of folding of the protein its being made to attach to

Question 8

how do we increase contrast of things when using electron microscopy

Answer 8

stain with heavy metal
these absorb e-
which shows up as dark, e- dense regions

Question 9

what can transmission EM be combined with

Answer 9

antibody labelling

Question 10

differential centrifugation: what’s the order of organelles that collect in the pellet after centrifuging

Answer 10

1 = nuclei
2= mitochndria
3 = ER and Golgi

remember, these are ‘enriched’ and not pure samples

Question 11

what does SDS stand for

Answer 11

sodium dodecyl sulphate

Question 12

how does SDS change proteins

Answer 12

gives them -ve charge
denatures them
they basically become -ve linear polypeptides that can then move through the polyacrylimide gel

Question 13

what is SDS-PAGE

Answer 13

way to visualise the proteins after electrophoresis via a bunch of diff stains