Lec 2 Crennell Flashcards
What is involved in the clarification of proteins?
- removing cell debris and other particulates
Once you have got rid of particulates and aggregates what must you not do to get rid of nucleic acids?
- don’t filter as DNA is fibrous and will clog filter device
Once you have got rid of particulates and aggregates what 3 ways can you get rid of nucleic acids?
- PRECIPITATE WITH 1% PROTAMINE SULPHATE
- Incubate with DNase
- extract with sonication
What is considered the best way to remove nucleic acids?
- sonication of proteins using shear chromosomes
- breaks open cells removing nucleic acids
Once you’ve removed nucleic acids, what 2 ways can you remove small molecules?
- ultrafiltration/buffer exchange
- dialysis
How does BUFFER EXCHANGE/ULTRAFILTRATION work?
Removal or small molecules
- put into centrifuge
- this allows small particles through but not large ones
- get rid of most small molecules if ran a few times
How does DIALYSIS work?
Removal or small molecules
- put into solution, equilibrates so lets out small molecules but not proteins
- keep replacing the buffer
You can purify your protein by putting it into E.Coli. What can happen though if you do this sometimes?
- e.coli can produce a large amount of the protein if you put it into it so you will end up with too much protein
- meaning you can’t fold them all at once
When purifying proteins, this is done so by exploiting physical and chemical properties. What are these 5 properties?
Charge - from sequence can work out charge
Hydrophobicity - most hydrophilic parts in centre, some on surface grouped together - cant predict where these are
Affinity - may have an idea if it binds metal ions
Solubility/Stability - depends on how strongly folded, and how large hydrophobic core, wouldn’t know unless you looked into this
Molecular Weight - need pure protein in order to calculate this
What way can you exploit solubility and what can this be used to purify?
- purification by precipitation
- protein or contaminants
Do you want to increase or decrease solubility of the protein when trying to precipitate it?
- Want to reduce solubility in order to precipitate other proteins
Precipitation SOLUBILITY
Changing ionic strength - What is salting in?
- first stage of the salting in salting out process
- low concentrations of salt is added to a protein solution
- the solubility increases
- Salt molecules stabilize protein
- no precipitation of the proteins
- increases solubility
Precipitation SOLUBILITY
Changing ionic strength - what is salting out?
- hydrophobic patches order H2O around them form water cages which are entropically unstable
- as salt conc increases from salting in concentration levels, H2O cages are removed exposing hydrophobic patches
- therefore proteins aggregate and precipitate out of solution
Process of salting out - in practice. What is used in the process?
Fractionation experiment -
- Add salt, centrifuge and separate precipitate, add salt/
- process repeats
Do the conditions of salting out have to be exothermic or endothermic and why?
- endothermic as you can’t have solution heating up
Precipitation SOLUBILITY
2) CHANGING SOLVENT - how does it work?
- Fractionation
- two charges on the protein, Q1 and Q2 on other.
- the charges are separated by a distance called R.
- They feel a force and that depends on what medium they’re sat in.
Precipitation SOLUBILITY
3) CHANGING pH - how does it work?
- At a pH close to proteins pI, it will be uncharged
- therefore may aggregate due to hydrophobic attraction
- this is known as ‘isoelectric precipitation’.
- may occur in dialysis
What can purification by exploiting stability only be used on and why?
- contaminants/impurites only (not protein)
- as its irreversible so denature impurities/contaminants only
Is purification by exploiting proteins on solubility by selective denaturation reversible or irreversible?
- IRREVERSIBLE
Apart from Solubility and Stability what do the other methods (CHAM) use?
Chromatography rather than fractionation/precipitation methods
What are the key features in chromatography which allow for it to work?
- stationary phase
- chromatographic bed eg column or glass or paper
- mobile phase
- delivery system
- detection system
- proteins separated by interaction with stationary phase
What is the stationary phase and how does it work?
- stationary phase which is thing which will interact with protein (immobilised on matrix)
- matrix: doesn’t interact, it is stable and inert.
What is high performance liquid chromatography?
- instead of a solvent, proteins forced through under high pressures
- much faster
- Incompressible stationary phases
- Continuous monitoring
How does chromatography actually work/what occurs?
- unbound proteins washed through
- bound proteins are eluted by a change in condition
- The peaks widen due to diffusion and retention on the column
- This limits the resolution of peaks
