8 + 9 - Methods in cell biology

Question 1

cytometry

Answer 1

characterize cell populations based on size, count, cell cycle, protein expression

Question 2

Cell fractionation

Answer 2

produce fractions enriched for certain cellular components by centrigugation

Question 3

how to generate contrast

Answer 3

contrast = important for visualizing details of a speciment

1. staining the specimen
2. using dark-field microscopy with oblique light source. Only light that is scattered by the specimen will be visible, the rest is aimed in another direction
3. phase contrast which changes the refractive index. The waves passing through the specimen will be out-of-phase with the other light
4. fluorescence microscopy with flourescent probes

Question 4

how to incorporate a fluorophore:

Answer 4

immunocytochemistry (Western Blot, primary Ab binds to the POI, secondary Ab with flouophore binds to primary Ab)

use GFP as marker, inject it into dna controlled by the same promoter as the GOI, place it at the end of the GOI so it is made along with the POI, fuse different flourescent proteins to two POIs, see if they interact (FRET, one flourecence exit will excite the other flourophore)

Question 5

Photoactivation

Answer 5

Involves synthetizing an inactive form of the fluorescent molecule of interest, introducing it to the cell, and then activating it suddenly at a chosen site in the cell by focusing a spot of light on it.

The technique allows us to follo wcomplex and rapid intracellular processes, such as the actions of signaling molecules or the movements of cytoskeletal proteins.
It is important that the POI remains biologically active despite being attached to a fluorophore. A bulky group can disrupt function.
Versions of GFP and others can also be made so that the flurorescence increases (or changes color) when hit by a strong light pulse. This allows the protein to fluorescence weakly during normal circumstances, and the scientist can follow the lifetime and behaviour of any protein

Question 6

FRAP

Answer 6

Fluorescence recovery after photobleaching

add flourophore to system, bleach one area of interest, see if the remaining flourophores wander into the area (and hw quickly)

Used to study:
movement and diffusion of proteins/molecules
connections between intracellular compartments
interactions of proteins with DNA