fractionation

Question 1

general dsp steps

Answer 1

1. Cell removal
2. Primary isolation
3. Purification
4. Final isolation

Question 2

macromolecules which can be separated out by centrifugation

Answer 2

Cells
• Subcellular components
• Proteins
• Nucleic acid

Question 3

Basis of centrifugal separation

Answer 3

. Size
.Shape
.Density

Question 4

disadvantages of bead mill for cell lysis

Answer 4

.temperature increases with bead volume
.poor scale-up
.high chance of contamination

Question 5

process variables of bead milling

Answer 5

.agitator speed
.proportion of beads
.bead size/type
.concentration of cell suspension
.flow rate

Question 6

Name methods of mechanical cell disruption

Answer 6

.bead mill
. rotor-stator mill
. high pressure
.ultra sonic vibration

Question 7

why is ultrasonic cell disruption not effective at large scales?

Answer 7

High energy requirements
Safety issue with noise
heating of sample
not continuous

Question 8

disadvantages of lysis via osmosis

Answer 8

1. low efficiency
2. may require enzymatic pretreatment
3. water usage is typically high
4. product dilution

Question 9

Name methods of chemical cell disruption

Answer 9

.osmosis
.enzyme digestion
.enzyme digestion
.solubilization
.lipid dissolution
.alkali treatment

Question 10

methods of DNA removal

Answer 10

1. precipitation
2. treat by beta-propiolactone ( i.e. in killed viral vaccine)
3. Adsorption depth filter
4. chromatographic methods
5. Nuclease treatment
6. Tangential flow filtration
7. Density gradient centrifugation

Question 11

what determines the solubility of a protein in a given solvent

Answer 11

The distribution of charge and
hydrophobic patches over the
surface of a protein,
which dictate the polar interactions with the aqueous solvent, ionic interactions with the salts present, and repulsive electrostatic forces between like-charged molecules.

Question 12

Factors that affect protein solubitlity ( which can be exploited to induce precipitation)exploited

Answer 12

.ionic strength
.pH
.dielectric constant
.temperature
. neutral salt addition

Question 13

effect of kosmotropic salt on solubility

Answer 13

wins water away, exposes hydrophobic patches, causing hydrophobic interactions between proteins and precipitation. decreases solubility.

Question 14

effect of chaotropic salt on solubility

Answer 14

promotes dissolution (water breakers), can cause proteins to denature