lecture 2 - purification by precipitation Flashcards
What contaminants does protein removal remove?
- particulates
- nucleic acids
- small molecules
- contaminating proteins from the expression host
Why is filtration not used to remove nucleic acids?
gelatinous/ clogs pores
What are the pros and cons of removing nucleic acids with protamine sulphate?
pro - quick
con - add contaminant, poor reproducibility , adding another peptide
What are the pros and cons of using DNase to remove nucleic acids?
- simple , reproducible
cons - slow, protease may degrade target
What is the best method for removing nucleic acids?
sanitation ( shears chromosomes)
How do you remove cell debris?
sedimentation viA centrifuging. Do not use filtration as it lowers the protein yield, as pores get clogged up with protein
How do you remove small molecules?
- buffer change
- dialysis, lets out small molecules ad not proteins. keep replacing the buffer on the outside, to get rid of all the small molecules
- ultrafiltration
What are the 5 properties of proteins that should be known so it can be isolated?
- Charge
- Hydrophobicity
- Affinity
- Solubility/ stability
- Molecular Weight
CHASM
What does ‘salting in’ do?
- increases ionic strength from below physiological
- stabilise the proteins and increases the solubility
What does ‘salting out’ do?
- removes water shielding from around the molecule by increasing the salt alot
- hydrophobic patches exposed on the protein
- promotes protein aggregation.
- ppt
How is salting out done in practice?
Add salt, centrifuge and separate ppt
Increase salt, ppt protein of interest
Remaining proteins in soluble fraction
What salt must be used in salting out?
- soluble , non - exothermic , pure, cheap and non interacting
What are the pros and cons of salting out?
pros -Limits bacterial growth
Prevents denaturation
Concentration step
cons - Need to desalt
Ineffectual if [protein] too low
Results vary with extract components
(co-precipitation)
What is the formula for precipitin by changing solvent?
F = q1 q2/ker^2
e= meant to represent the dielectric constant
r is the separation
k is a constant
What happens when you add organic solvents to a solution?
- organic solvent lies over the hydrophobic sections
- makes these parts very solvated
- lowers the dielectric constant but increases electrostatic attraction causing ppt
What is a problem with precipitation by adding organic solvent?
organic solvent may inactivate the protein
What happens when a protein has a pH that is similar to its pI/ IEP?
A protein will be uncharged and may aggregate due to hydrophobic attraction
What does exploiting stability do?
- purification by selective denaturation
- contaminants only
How can you precipitate extremophiles?
denaturation by temp , pH and solvent
What are the 5 key components of chromatography equipment?
1 - stationary phase 2 - chromatographic bed 3 - mobile phase 4 - delivery system 5 - detection system
What is the stationary phase made up of?
a matrix - stable , inert , high capacity
What are 4 key features of High Performance Liquid Chromatography?
- Incompressible stationary phases
- Pumps (High Pressure and Flow Pressure FPLC)
- Rapid separation
- Continuous monitoring
What is the chromatography resolution equation?
R = separation/ average width
R = 2S / W1+W2
What do different peaks represent on a chromatography graph?
unbound comes off earliest , is the first peak
How does isoelectric precipitation by changing the ph work?
At a pH close to its pI , a protein will be uncharge and may aggregate , due to its hydrophobic attraction. This is isoelectric precipitation
What limits the resolution of chromatography peaks?
- The peaks widen due to diffusion and retention on the column
What salt is used for salting out?
(NH4)2SO4 saturated at 4M
What is an overview of protein purification?
- preparation, extraction and clarification
- Capture
- Intermediate purification
- Polishing
What does the clarification stage do?
Removes cells debris and other particulates
What are the techniques used for clarification?
- filtration ( can lower protein yield)
- Sedimentation ( iva centrifugation on a large or small scale )
What are the 3 ways that solubility can be exploited to purify by precipitation?
- ) Changing the ionic strength ( salting in and salting out)
- )changing the solvent
- ) changing the pH
What does S represent in the chromatography equation?
S Is the separation of the maxima
What does W1 and W2 represent in the chromatography equation?
W represents the widths
