1. Exam next week: Chapter 4?
Lab next week
Today: protein purification/general strategies

2. Protein purification: Separate relevant proteins from all other cellular proteins

3. Chromatographic separation of proteins
Exploit differences in physical/chemical characteristics
Charge
Size (effective size)
Ligand binding (ability to form strong interactions with other molecules)
Hydrophobicity

4. Chromatography
Typically: add a mixture in a mobile phase to some sort of stationary phase
Some components have affinity for the mobile phase, some for the stationary phase
Mobile and stationary phase separate

6. Protein mixture (A,B,C, other) in mobile phase
Proteins have different
affinities for stationary/mobile
phases
Proteins elute at different times
spectrophotometer

7. Chromatogram: Two pure proteins
3-7 minutes
10-15 minutes
A280
Retention time

8. Types of protein chromatography
Ion-exchange
Cation
Anion
Size-exclusion
a.k.a. Gel filtration
Affinity

9. Affinity chromatography
Stationary phase: ligand tightly bound to an insoluble ‘resin’
Protein of interest has an affinity for the ligand
‘Natural’ affinity: eg. an ATP-binding protein
‘Artificial’ affinity: eg. use genetic manipulation to add a binding site

10. Ion exchange chromatography
Mobile phase: aqueous
Important variables:
Salt concentration pH
Stationary phase:
Insoluble “bead” with covalently-bound, charged group

11. Examples of ion exchange resins+
Cl-
Quaternary amine
Insoluble
polymer
Ion
exchanger
Counter-ion O - S O
Na+
Sulfonate O

12. Ion exchange Cation exchange Anion exchange
Matrix is negatively charged
Counter-ions are cations
Selective binding of positively-charged proteins
Anion exchange
Matrix is positively charged
Counter-ions are anions
Selective binding of negatively-charged proteins

13. Anion exchange - + Counter-ions: anions (neg. charge) + +
Cl-
Cl- +
Proteins with net negative charge
have affinity for stationary phase
“Exchange” of anions on resin
Proteins with net positive charge have
relatively higher affinity for mobile phase +
Cl-
Cl-
Cl- + + +

14. Cation exchange The higher the positive charge, the longer a
protein takes to elute

15. Changing of the mobile phase
Low to high salt concentration
Protein of interest has net positive charge
Strong adherence to stationary phase (cation exchange)
“Stuck” on the column
Increase salt concentration: decrease protein’s affinity for stationary phase
Move protein to mobile phase & recover it

16. Anion Exchange Chromatography
Anionic (negatively charged)
proteins adhere to a positively
charged resin
‘Exchange’ occurs when
another source of anions
(eg. salt) is introduced
Proteins with different charge
characteristics elute (become
‘unstuck’) at different salt
concentrations
‘Separation’ of proteins is
achieved because different
proteins (1) don’t stick at all,
(2) stick weakly, or (3) stick
strongly depending on their
biophysical characteristics

17. Gel filtration/Size exclusion: Separation based on “size”
Very large proteins (or protein complexes or other molecules) traverse the column quickly [elute first]
Very small proteins have the largest accessible volume and the farthest to travel: traverse the column very slowly [elute last]
Proteins/complexes between these extremes traverse the column at varying speeds

18. Gel Filtration/Size Exclusion
Chromatography
resin (stat. phase)
Polymer beads with
tiny ‘pores’

19. Gel Filtration/Size Exclusion

20. GF: Separation based on size