Isolating and analysing proteins Flashcards
Biologics
Protein-based drugs, monoclonal antibodies, therapeutic proteins (insulin), vaccines.
Salting in/out of proteins
Solubility depends on salt concentration (dependent on protein charge), low salt aggregates, high salt no interaction with water, most stabile in the middle (salt screening). Ammonium sulphate precipitation to isolate natural proteins.
Chromatography
column contains stationary phase (beads/resin), mixture sits on top and buffer is dripped through as mobile phase. Some molecules interact better with the stationary phase and will separate based on affinity for both the mobile and stationary phase.
Size exclusion CT (gel filtration)
Stationary - porous beads, mesh, cellulose
Separates by size, small molecules enter beads, large are too big. Small beads get stuck in there and move slower. Like walking through a mall. largest beads leave first to estimate the molecular weight based on the RETENTION VOLUME.
Ion exchange chromatography
Stationary phase - to bind anion (net +), to bind cation (net -), charge on resin.
Direct binding to beads based on charge interactions. Elute the protein by changing the buffer pH, salt concentration to (weaken) screen charge-charge interactions.
Affinity CT
Stationary - contains ligand for protein.
Immobilise certain protein and wash away the rest, then elute using a ligand to compete.
Purest samples are produced through
Affinity chromatography
Affinity tags
Matrix bound ligand, protein of interest hold affinity tag to bind ligand. Recombinant protein may be produced with the tag to purify ANY PROTEIN.
Common affinity tag process
6x histidine, coordinate Ni+ to bind specifically bind resin. Imidazole elutes to compete for the nickel of the stationary phase, kicking the protein off.
SDS-PAGE
- Incubate protein with SDS and reducing agent (β-mercaptoethanol breaks disulfide bonds) to denature by coating in negative charge.
- Separate with electric field to pull towards + down.
- determines size and purity.
2D page
Isolate by pI and then by size, spread out, then pull down.
- reveals disease related differences in protein levels.
X-ray crystallography
Measures diffraction pattern of crystallised protein to determine the 3D arrangement of atoms, lots of math ensues. Fit atomic model to electron positions.
Cryo-electron microscopy
Taking a picture using electrons, must be cool to not destroy the protein. Like a camera that can be reconstructed to create 3D structure similar to a CT scan. we can see atoms!!! protein can be in solution, no need for crystallisation.
NMR
Superconducting magnets to measure magnetic environments of the nuclei in a protein. Determine which are close together and the structure AND motion. Computer modelling.
Done at room temp (liquid buffer), biologically relevant.
3D structure of Aβ(1-42) fibrils in alzheimers
We know the structure of the plaques, amyloid aggregation gives us data to work on and target the hydrophobic interface with drugs.
