lecture 4 - purification by solubility

Question 1

definition of purification

Answer 1

to remove unwanted material and maximize recovery of the target protein

Question 2

what properties can we use (5)

Answer 2

solubility, charge, hydrophobicity, size, affinity to a specific ligand

Question 3

purification by solubility depends on:

Answer 3

self-self interactions

Question 4

solubility differs due to:

Answer 4

different amino acids composition of surface residues

Question 5

properties that affect solubility (3)

Answer 5

(1) charge (pH vs pI) - surface of protein (where ineractions are) will have some charges
(2) concentration of salt (usually NO4, SO4)
(3) polarity/hydrophobicity

Question 6

can separate molecules by solubitiy using

Answer 6

centrifugation and max point of solubility for a protein (think solubility graph)

Question 7

at a low salt concentration

Answer 7

• charges may be localized in patches if there are sufficient attractive interactions
• aggregation
• causes precipitation (decreased solubility)

Question 8

if add some salt to a solution with low salt concentration

“salting in”

Answer 8

• salt ions interact with opposite charges on protein (NH4+ near Asp-)
• sheilds protein charges from interacting with each other
• protein charges are neutralized
• theres a decrease in attractive reactions
• theres an increase in solubility

Question 9

if add a lot of salt to a solution with some salt

“salting out”

Answer 9

• salt ions tie up h2o - reducing h2o available to hydrate protein
• enhances hydrophobic effect, increasing aggregation via hydrophobic patches on protein surface
• decreases solubility

Question 10

effect of pH >/< pI on solubility

Answer 10

• results in electrostatic repulsion

- increases solubility

Question 11

effect of pH = pI on solubility

Answer 11

• no net charge
• no interactions
• decrease in solubility

Question 12

pH > pI (net charge?)

Answer 12

net neg charge

Question 13

pH < pI (net charge?)

Answer 13

net pos charge

Question 14

e.g. explain how can add salt to get protein of interest

Answer 14

(1) add salt to cell extract to get ppt 1 and sup 1
(2) add 50% salt to sup 1 to get ppt 2 and sup 2
(3) examine ppt 1, ppt2, sup2 for presence of protein of interest and for contaminating proteins

Question 15

how does column chromatography work?

based on what interactions? what do these interactions do?

Answer 15

• based on interaction of proteins with stationary (s) phase
• these interactions impede mobility
• more time in mobile (l) phase = faster elution
• liquid added to column, flows continuously through the stationary phase, elutions collected in equal fractions.

Question 16

how check fractions from chromatography for presence of protein?

Answer 16

• assay

- if come out later they interact more with the stationary phase