Lecture 2

Question 1

‘Purification’

Answer 1

removing other proteins

Question 2

Most common contaminants once cell lysis?

Answer 2

Particulates and nucleic acids

Question 3

Filtration can lower??

Answer 3

Protein yield

Question 4

Supernatant?

Answer 4

Soluble proteins

Question 5

Pellet usually consists of ?

Answer 5

Membrane fractions, organelles

Question 6

Why can’t we filter nucleic acids out?

Answer 6

• Fibrous (nucleic acids)
• Filter filtration devices
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Question 7

How does protamine sulphate remove nucleic acids?

Answer 7

positive peptide and therefore interacts with DNA (neg) and precipitates it

Question 8

Pros and cons with protamine sulphate?

Answer 8

cons: Needs to be purified out afterwards, which leads to this method having a poor reproducibility
pros: rapid and simple

Question 9

How else can we remove nucleic acids from a solution (other than protamine sulphate and sonnication ). Issue with this?

Answer 9

DNAse (25-37 degrees) for 30 minutes

slow

Question 10

sonnication method effect on nucleic acids?

Answer 10

shears chromosome

Question 11

Ultrafiltration works by which two concepts??

Answer 11

Semi-permeable membranes

define MW cutoff

Question 12

Dialysis method?

Answer 12

In solution
Pores let out small molecules and not proteins (large)
Replace buffer on outside to ensure small amount of small molecule left
(ensure the concentration gradient is large)

Question 13

CHASM?

Answer 13

C- charge (isoelectric point/surface charge)
H- hydrophobicity (most in the hydrophobic core, some patches)
A- affinity (Activity, metal ions, enzyme?/Substrate)
S-solubility ( how large is the hydrophobic core)
M-Molecular weight (does not include post translational modifications)

Question 14

How do we calculate surface charge?

Answer 14

worked out from experimentation

Question 15

Isoelectric point?

Answer 15

point at which the internal charge of a molecule is balanced

Question 16

Stability of the protein depends on??

Answer 16

How large the hydrophobic core is

Question 17

solubility determines what?

Answer 17

Interaction between proteins

Question 18

counterbalance acidic and basic residues????

Answer 18

Surrounded by ions

Question 19

Hydrophobic patches surrounded by

Answer 19

Water molecules which face away

Question 20

How can we exploit solubility in order to induce precipitation?

Answer 20

Reduce solubility in order to precipitate selectivelyCan do this by changing charge and water that is available within the solution

Question 21

Physiological ionic strength?

Answer 21

0.15-0.2M

Question 22

Way in which we can increase ionic strength?

Answer 22

Add salt

Question 23

At a low salt conc?

Answer 23

proteins have lower solubility

Question 24

At a high salt conc?

Answer 24

proteins have higher solubility

Question 25

At a med salt conc?

Answer 25

More soluble

Question 26

Salting out method works by?

Answer 26

Forming water cages (at peak solubility), decreases entropy (water molecules become more organised), Excess salt attracts water away from the entropy cage and it reduces in size (decreasing solubility again)

Question 27

Interaction increases ______ in salting out

Answer 27

aggregation

Question 28

Proteins with more and larger hydrophobic patches?

Answer 28

Salt out faster e.g haemoglobin has a high ionic strength as it requires more salt in order to causes salting out this is due to its location being within the blood

Question 29

Fractionation experiment?

Answer 29

Add salt, centrifuge, separate precipitate. increase salt, ppt protein of interest remaining (supernatant)

Question 30

Issues with salting out in practice?

Answer 30

Trial and error, may be time consuming See where the protein precipitates have to desalt

Question 31

In the first step of salting out what will first fall to the bottom after centrifugation?

Answer 31

Most hydrophobic protein

Question 32

How to select for a suitable salt, eg ???

Answer 32

soluble, non-exothermic, pure, cheap, non-interacting (NH4)2SO4, saturated at 4M

Question 33

Why does a salt need to be non exothermic ?

Answer 33

need to keep temp low for proteins), if it heats up, denaturation

Question 34

Salting out:cons

Answer 34

- Need to desalt - Ineffectual if [protein] too low (small volumes eof protein won't precipitate as well as they don't meet the molecules they are to aggregate with) - Results vary with extract components (co-precipitation: some molecules when they precipitate bring down others with them )

Question 35

Salting out: pros

Answer 35

Limits bacterial growth Prevents denaturation Concentration step

Question 36

What is effected when we change the solvent?

Answer 36

Charges will feel different repulsion or attraction depending on the distance at which they are kept.

Question 37

Screening effect of two charges held within a solvent

Answer 37

K- dielectric constant

Question 38

by changing the solvent we change the

Answer 38

Solvating power

Question 39

E (weird greek e) =

Answer 39

Solvating power

Question 40

forces between two charges equation?

Answer 40

FBTC=( KxQ1xQ2)/ Er^2 k- dielectric constant q1 and q2 are the charges of two molecules e- solvating power r- distance between the two charges

Question 41

What causes precipitation when changing the solvent?

Answer 41

Electrostatic interactions

Question 42

Decreasing E increases ?...

Answer 42

Increases electrostatic attraction

Question 43

By adding organic solvents

Answer 43

Get rid of hydrophobic patches solvated lower the dielectric contrast therefore electrostatic attraction increases= precipitation

Question 44

E of organic solvents vs water

Answer 44

OS: 1-10 Water: 80

Question 45

Issue with adding inorganic solvents to separate

Answer 45

may cause inactivation of your protein of interest (unfolding)

Question 46

pI

Answer 46

isoelectric point

Question 47

Shape of curve in which pH increases for a protein and the measure of its solubility

Answer 47

At its pI, solubility at its minimum | upside down bell

Question 48

At pI protein molecules?

Answer 48

Attract each-other

Question 49

When pH>pI

Answer 49

protein molecules repellants eachother

Question 50

when pH < pI

Answer 50

also repellants eachother