Protein Purification Flashcards
What is protein purification?
Separation of proteins to access the protein you want from all the others In the preparation
How to tell if a protein is pure?
Use analytical methods that can separate proteins with high resolution
SDS page
SDS = detergent
Take chemical acrylamide and polyrise to make gel
SDS unfolds the protein by coating unfolded chain
Place proteins into gel and run electrical current (compare to known marker)
Proteins will separate by molecular mass: smaller will move further
Not useful for studying protein structure as have to denature them for it to work but can work out size, quantity and type of protein.
Separation by solubility
Surface of soluble proteins feature many polar residues = solubility differs because surface differs
Interact with water to keep in solution
Most common method of separating by solubility
Precipitation by ammonium sulphate (salting out)
Depends on polarity of protein surface: determines how soluble it is in water.
Dissolved salt needs to be solvated so adding ammonium sulphate decreases the water available to solvate the protein
Add 1st concentration of ammonium sulphate then centrifuge, some proteins will drop out. Will have pellet and precipitate then separate and keep going until have desired proteins seperated.
Isoelectric focusing
Isoelectric point = PH where molecule has no net charge
Uses specially constructed strips with PH gradients on them.
Take strips and place in electric field then add proteins
Is proteins negatively charges it will be drawn towards positive side
As it moves along the strip the PH will change until point where neutral net charge
Protein focused at that point
Stain strip with coomassie blue or silver nitrate and bands of protein revealed
Two dimensional gel
Do isoelectric focusing and embed at top of SDS page gel and run at 90 degrees to IEF
Separates on the basis of 2 properties = much better resolution and stops multiple proteins being in one band
How to observe proteins
Most proteins colourless but absorb UV light at 280nm
Due to aromatic amino acid residues
Size exclusion chromatography
In Colum pour resin beads and then pour proteins through the beads: the beads are made of cross linked structure with lots of tiny pockets.
Small proteins get stuck in pockets but large proteins bounce back out = takes small proteins longer to pass through then the large proteins the larger the protein the quicker it passes through.
How can you use size exclusion chromatography to separate proteins
Can use to estimate the type of protein
Take proteins of a known size and run through the coloum measuring the volume you collected before it emerged.
Then create a graph to estimate the protein.
However Could be the same protein with a different structure and will still separated differently due to mass difference
Ion exchange chromatography
Depends on ionisable groups on the proteins surface
Pour into Colum with resin beads covered in charged groups
Pos charges interact with protein neg residues and bond
Causes proteins to shift PH. Then add salt in a gradient.
Weakly bound proteins leave Colum first
Affinity chromatography
Most proteins interact with something in a specific way e.g. enzyme with substrate
Get matrix in Colum that’s decorated with enzymes specific molecule
Pour proteins through and desired protein will bind whilst others wont.
Protein then has to be recovered:
- could shift PH by adding salt
- most common: add unbound free ligand and protein will unbind from matrix and bind to that instead
Expression and affinity purification of fusion protein
If dont know protein:
Take DNA and place into bacterium = bacterium will replicate
Add gene of a different protein to front of protein gene = fusion protein
If added bit has specific affinity for ligand can use that ligand to separate it
