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BB10186 > Lecture 2 > Flashcards

Lecture 2 Flashcards

1
Q

‘Purification’

A

removing other proteins

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2
Q

Most common contaminants once cell lysis?

A

Particulates and nucleic acids

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3
Q

Filtration can lower??

A

Protein yield

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4
Q

Supernatant?

A

Soluble proteins

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5
Q

Pellet usually consists of ?

A

Membrane fractions, organelles

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6
Q

Why can’t we filter nucleic acids out?

A
  • Fibrous (nucleic acids)
  • Filter filtration devices
    `
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7
Q

How does protamine sulphate remove nucleic acids?

A

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

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8
Q

Pros and cons with protamine sulphate?

A

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

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9
Q

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

A

DNAse (25-37 degrees) for 30 minutes

slow

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10
Q

sonnication method effect on nucleic acids?

A

shears chromosome

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11
Q

Ultrafiltration works by which two concepts??

A

Semi-permeable membranes

define MW cutoff

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12
Q

Dialysis method?

A

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)

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13
Q

CHASM?

A

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)

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14
Q

How do we calculate surface charge?

A

worked out from experimentation

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15
Q

Isoelectric point?

A

point at which the internal charge of a molecule is balanced

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16
Q

Stability of the protein depends on??

A

How large the hydrophobic core is

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17
Q

solubility determines what?

A

Interaction between proteins

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18
Q

counterbalance acidic and basic residues????

A

Surrounded by ions

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19
Q

Hydrophobic patches surrounded by

A

Water molecules which face away

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20
Q

How can we exploit solubility in order to induce precipitation?

A

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

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21
Q

Physiological ionic strength?

A

0.15-0.2M

22
Q

Way in which we can increase ionic strength?

A

Add salt

23
Q

At a low salt conc?

A

proteins have lower solubility

24
Q

At a high salt conc?

A

proteins have higher solubility

25
Q

At a med salt conc?

A

More soluble

26
Q

Salting out method works by?

A

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)

27
Q

Interaction increases ______ in salting out

A

aggregation

28
Q

Proteins with more and larger hydrophobic patches?

A

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

29
Q

Fractionation experiment?

A

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

30
Q

Issues with salting out in practice?

A

Trial and error, may be time consuming

See where the protein precipitates

have to desalt

31
Q

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

A

Most hydrophobic protein

32
Q

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

A

soluble, non-exothermic, pure, cheap, non-interacting

(NH4)2SO4, saturated at 4M

33
Q

Why does a salt need to be non exothermic ?

A

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

34
Q

Salting out:cons

A
  • 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 )
35
Q

Salting out: pros

A

Limits bacterial growth
Prevents denaturation
Concentration step

36
Q

What is effected when we change the solvent?

A

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

37
Q

Screening effect of two charges held within a solvent

A

K- dielectric constant

38
Q

by changing the solvent we change the

A

Solvating power

39
Q

E (weird greek e) =

A

Solvating power

40
Q

forces between two charges equation?

A

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

41
Q

What causes precipitation when changing the solvent?

A

Electrostatic interactions

42
Q

Decreasing E increases ?…

A

Increases electrostatic attraction

43
Q

By adding organic solvents

A

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

44
Q

E of organic solvents vs water

A

OS: 1-10
Water: 80

45
Q

Issue with adding inorganic solvents to separate

A

may cause inactivation of your protein of interest (unfolding)

46
Q

pI

A

isoelectric point

47
Q

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

A

At its pI, solubility at its minimum

upside down bell

48
Q

At pI protein molecules?

A

Attract each-other

49
Q

When pH>pI

A

protein molecules repellants eachother

50
Q

when pH < pI

A

also repellants eachother

BB10186 (6 decks)

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