Protein Purification

Question 1

How are molecules separated in gel filtration?

Answer 1

Molecules in solution are separated by size

Question 2

What size of molecule will be eluted first?

Answer 2

Large molecules will be eluted first as they will be absorbed by less of the pores

Question 3

What is the alternative name for gel filtration chromatography?

Answer 3

Size exclusion chromatography

Question 4

What is the void volume equivalent to? (V0)

Answer 4

the molecules which were excluded from the beads, which is equivalent to the volume of the liquid outside the beads

Question 5

What is the whole volume of the liquid in the column equivalent to?

Answer 5

VI = the elution volume for the smallest molecules

Question 6

What is the total column volume (Vt)

Answer 6

Vt=Vo+Vi+Vs (the volume of liquid outside the beads plus the volume inside the beads plus the volume of the beads themselves)

Question 7

What is KAV equal to?

Answer 7

KAV=(Ve-Vo)/(Vt-Vo)

Question 8

What is KAV proportional to?

Answer 8

The volume inside the beads available to a given molecule, independent of the volume used

Question 9

What do you plot KAV against in a calibration plot?

Answer 9

Log of MW

Question 10

What are the three most common practical applications of gel filtration?

Answer 10

1. Desalting or buffer exchange
2. Purification of macromolecules
3. Analysis of oligomeric states of proteins and protein complexes

Question 11

How can you use gel filtration to analyse the oligomeric state of a protein?

Answer 11

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

Question 12

What type of gel matrix is used for desalting of proteins?

Answer 12

Media containing beads with small pores (eg Sephadex G28)

Question 13

What would be the difference between a fibrous or a globular protein travelling down a gel filtration column?

Answer 13

Globular proteins will fit more easily into pores, whereas fibrous proteins wouldn’t fit aswell so might have an increased apparent Molecular Weight

Question 14

What is a different technique you could use to check the accuracy of your gel column results?

Answer 14

Dynamic Light Scattering

Question 15

What are the basic principles of SDS Page?

Answer 15

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

Question 16

What does Ion Exchange Chromatography separate proteins by?

Answer 16

Separates proteins according to their net surface charge

Question 17

What is the other term for the net surface charge exploited in IEC?

Answer 17

Isoelectric point (Pl is pH at which the net charge = 0)

Question 18

At which set of isoelectric points are proteins considered acidic

Answer 18

Pl<7

Question 19

What type of proteins are separated by anion exchange?

Answer 19

negatively charged proteins - captured by positively charged matrices

Question 20

What type of proteins are separated by cation exchange?

Answer 20

Positively charged proteins are captured by negatively charged matrix

Question 21

How does Hydrophobic Interaction Chromatography separate proteins?

Answer 21

Separates proteins due to hydrophobic interactions (assuming hydrophobic residues are exposed to some extent on the protein surface)

Question 22

What condition is required for HIC?

Answer 22

water must be removed from protein surface (you can use high salt concentrations)

Question 23

How can you elute proteins from HIC column?

Answer 23

Wash with low salt buffer

Question 24

What makes affinity chromatography different from other types of chromatography?

Answer 24

Separates proteins from engineered specific state (eg Fusion TAG protein) whereas other chromatographies exploit intrinsic properties of the properties

Question 25

What are the two most common TAGS used in affinity chromatography?

Answer 25

His6 or GST

Question 26

How does ligand chromatography differ from standard affinity chromatography?

Answer 26

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)

Question 27

What is used to create Dye Chromatography matrices?

Answer 27

synthetic polycyclic dyes used to create ligands with structural similarities to cofactors eg NADH (High salt used to elute proteins none specifically)

Question 28

What matrix would you use to elute a fusion tag protein with His6?

Answer 28

An agarose matrices containing Nickel or Cobalt