electrophoresis

Question 1

define buffer capacity

Answer 1

the extent of resistance to buffer change

Question 2

Why does a desirable buffer have low diffusion in water, and pH around operating pH

Answer 2

Buffer dictates pH. Want low diffusion in water so that pH remains constant and doesn’t concentrate in areas. Buffer concentration implies more ions which generates more current and hence current, potentially leading to boiling

Question 3

What does electrophoresis mobility depend on?

Answer 3

1. What the molecule is moving through
2. the voltage gradient
3. viscosity of the solution
   (amongst other things)

Question 4

Considerations when scaling electrophoresis

Answer 4

increase the area and keep voltage the same
requires a large power supply ( cost)
limiting factor= temperature ( more current more heat)
Heat damages circuit and protein

Question 5

Key limiting parameters of electrophoresis

Answer 5

Resistance established over the length of the process ( the longer the length, the higher the resistance)
electrode material

Question 6

define ampholine

Answer 6

a combination of small zwitterionic compound which overpower the local effect on pH of compound being seperated and retain a constant pH gradient. may be removed using dialysis

Question 7

strengths of MACS

Answer 7

gently
easily combined with other metods
fast
carry out positive and negative selection
reproducible
easy to scale

Question 8

weaknesses of MACS

Answer 8

labour intensive
only purify one popn at a time
not effective if popn is already <90% pure
beads are expensive
single use column
beads can damage delicate cells
yield loss due to multiple centrifuge spins during binding

Question 9

Functioning of FACS

Answer 9

fluorescently labels proteins
cells flow through a narrow channel
fluorescence measured by a laser
droplets are charged according to fluorescence
droplets are collected according to assigned charge using deflector plates

Question 10

FACS strengths

Answer 10

.versatile
. multiple sorts in one go
.gives high purity
.High throughput
.sensitive
.takes into account expression, size and granularity

Question 11

FACS weaknesses

Answer 11

.difficult to operate
.slow
.miltilazer interaction
.expensive equipment
. requires high number of cells
mistakes occur if cells are too small or flow too fast
.cells can suffer shear stress or electrically induced damage

Question 12

requirement for mammalien cell seperation

Answer 12

maximise yield, retain viability of cells and sterility