Flashcards in Lecture 2 Deck (50)
removing other proteins
Most common contaminants once cell lysis?
Particulates and nucleic acids
Filtration can lower??
Pellet usually consists of ?
Membrane fractions, organelles
Why can't we filter nucleic acids out?
- Fibrous (nucleic acids)
- Filter filtration devices
How does protamine sulphate remove nucleic acids?
positive peptide and therefore interacts with DNA (neg) and precipitates it
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
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
sonnication method effect on nucleic acids?
Ultrafiltration works by which two concepts??
define MW cutoff
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)
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)
How do we calculate surface charge?
worked out from experimentation
point at which the internal charge of a molecule is balanced
Stability of the protein depends on??
How large the hydrophobic core is
solubility determines what?
Interaction between proteins
counterbalance acidic and basic residues????
Surrounded by ions
Hydrophobic patches surrounded by
Water molecules which face away
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
Physiological ionic strength?
Way in which we can increase ionic strength?
At a low salt conc?
proteins have lower solubility
At a high salt conc?
proteins have higher solubility
At a med salt conc?
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)
Interaction increases ______ in salting out
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
Add salt, centrifuge, separate precipitate.
increase salt, ppt protein of interest