Protein Purification Flashcards
How are molecules separated in gel filtration?
Molecules in solution are separated by size
What size of molecule will be eluted first?
Large molecules will be eluted first as they will be absorbed by less of the pores
What is the alternative name for gel filtration chromatography?
Size exclusion chromatography
What is the void volume equivalent to? (V0)
the molecules which were excluded from the beads, which is equivalent to the volume of the liquid outside the beads
What is the whole volume of the liquid in the column equivalent to?
VI = the elution volume for the smallest molecules
What is the total column volume (Vt)
Vt=Vo+Vi+Vs (the volume of liquid outside the beads plus the volume inside the beads plus the volume of the beads themselves)
What is KAV equal to?
KAV=(Ve-Vo)/(Vt-Vo)
What is KAV proportional to?
The volume inside the beads available to a given molecule, independent of the volume used
What do you plot KAV against in a calibration plot?
Log of MW
What are the three most common practical applications of gel filtration?
- Desalting or buffer exchange
- Purification of macromolecules
- Analysis of oligomeric states of proteins and protein complexes
How can you use gel filtration to analyse the oligomeric state of a protein?
Column can be calibrated using proteins of a known size (MW) - make calibration plot and can subsequently used to determine the size of protein in question
What type of gel matrix is used for desalting of proteins?
Media containing beads with small pores (eg Sephadex G28)
What would be the difference between a fibrous or a globular protein travelling down a gel filtration column?
Globular proteins will fit more easily into pores, whereas fibrous proteins wouldn’t fit aswell so might have an increased apparent Molecular Weight
What is a different technique you could use to check the accuracy of your gel column results?
Dynamic Light Scattering
What are the basic principles of SDS Page?
Denature proteins and heat with anionic detergent SDS
The proteins will be coated with a negative charge from the SDS - will have a uniform charge density.
Proteins travel towards the electrode - proteins with larger MW will move slower and therefore less far in the Gel
What does Ion Exchange Chromatography separate proteins by?
Separates proteins according to their net surface charge
What is the other term for the net surface charge exploited in IEC?
Isoelectric point (Pl is pH at which the net charge = 0)
At which set of isoelectric points are proteins considered acidic
Pl<7
What type of proteins are separated by anion exchange?
negatively charged proteins - captured by positively charged matrices
What type of proteins are separated by cation exchange?
Positively charged proteins are captured by negatively charged matrix
How does Hydrophobic Interaction Chromatography separate proteins?
Separates proteins due to hydrophobic interactions (assuming hydrophobic residues are exposed to some extent on the protein surface)
What condition is required for HIC?
water must be removed from protein surface (you can use high salt concentrations)
How can you elute proteins from HIC column?
Wash with low salt buffer
What makes affinity chromatography different from other types of chromatography?
Separates proteins from engineered specific state (eg Fusion TAG protein) whereas other chromatographies exploit intrinsic properties of the properties
What are the two most common TAGS used in affinity chromatography?
His6 or GST
How does ligand chromatography differ from standard affinity chromatography?
Ligand chromatography uses a matrix containing substrates very similar to the natural substrate or native ligand of the protein - (cross linked heparin used to bind DNA)
What is used to create Dye Chromatography matrices?
synthetic polycyclic dyes used to create ligands with structural similarities to cofactors eg NADH (High salt used to elute proteins none specifically)
What matrix would you use to elute a fusion tag protein with His6?
An agarose matrices containing Nickel or Cobalt
