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1

'Purification'

removing other proteins

2

Most common contaminants once cell lysis?

Particulates and nucleic acids

3

Filtration can lower??

Protein yield

4

Supernatant?

Soluble proteins

5

Pellet usually consists of ?

Membrane fractions, organelles

6

Why can't we filter nucleic acids out?

- Fibrous (nucleic acids)
- Filter filtration devices
`

7

How does protamine sulphate remove nucleic acids?

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

8

Pros and cons with protamine sulphate?

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

9

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

DNAse (25-37 degrees) for 30 minutes
slow

10

sonnication method effect on nucleic acids?

shears chromosome

11

Ultrafiltration works by which two concepts??

Semi-permeable membranes
define MW cutoff

12

Dialysis method?

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)

13

CHASM?

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)

14

How do we calculate surface charge?

worked out from experimentation

15

Isoelectric point?

point at which the internal charge of a molecule is balanced

16

Stability of the protein depends on??

How large the hydrophobic core is

17

solubility determines what?

Interaction between proteins

18

counterbalance acidic and basic residues????

Surrounded by ions

19

Hydrophobic patches surrounded by

Water molecules which face away

20

How can we exploit solubility in order to induce precipitation?

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

21

Physiological ionic strength?

0.15-0.2M

22

Way in which we can increase ionic strength?

Add salt

23

At a low salt conc?

proteins have lower solubility

24

At a high salt conc?

proteins have higher solubility

25

At a med salt conc?

More soluble

26

Salting out method works by?

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

Interaction increases ______ in salting out

aggregation

28

Proteins with more and larger hydrophobic patches?

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

Fractionation experiment?

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

30

Issues with salting out in practice?

Trial and error, may be time consuming

See where the protein precipitates

have to desalt