Professional Documents
Culture Documents
and chromatography
1. Electrophoresis
a Sketch an electrophoresis system.
Derive the electrophoretic mobility as a function of the Stokes radius and charge, and discuss their
effects on the mobility.
Homepage: http://aula.au.dk/courses/AULA100407093252/
Page 1/3
g
h
i
j
The hydrophobic tail of SDS breaks up the hydrophobic core, unfolds structured regions and breaks
complexes. Long rodlike complexes with a charge proportional to the Mw are formed.
How does the charge and hydrodynamic radius depend on the size of the protein in an SDS page gel,
and what is the effect of the size on the electrophoretic mobility?
Both increase linearly with the MW. The electrophoretic mobility decreases.
Where does the separation come from in SDS page?
Separation is due to increased friction from the interaction with the gel.
How do you judge the size of a protein from and SDS page gel?
From a comparison with molecular weight standards (proteins with known MW).
If a protein is not really unfolded by SDS would it then run as a larger or smaller protein, and why so?
Smaller since the hydrodynamic radius is smaller.
Describe how you run a 2D gel.
First do isoelectric focusing and then run an SDS gel in the orthogonal direction.
2. Liquid chromatography
a Sketch a liquid chromatography system.
3. Affinity chromatography
Describe the ligands used, binding, and elution when purifying
a a His-tagged protein
b an enzyme
c Why might one want to use a spacer?
Ligand
Elution
a
Ni, Zn
Imidazol, EDTA, acid
b
Substrate analog
Substrate analog, substrate
c For the ligand to be able to reach into the active site without being hindered by the matrix.
4. Ion exchange chromatography
In cation exchange chromatography, positively charged molecules are retained because the stationary
phase displays a negatively charged functional group, and in anion exchange chromatography negatively
charged molecules are retained because the stationary phase displays a positively charged functional group.
a Out of three proteins (A, B and C) with pI = 3, 5 and 7, which are likely to bind to cation and anion
exchange columns with running buffers at pH 5, 7 and 8?
Homepage: http://aula.au.dk/courses/AULA100407093252/
Page 2/3
b
c
d
pH
cation
anion
5
C
A
7
A, B
8
A, B, C
Which of the three proteins are separable from the rest in one ion exchange step, and how?
A and C, e.g. with cation and anion respectively exchange at ph 5.
Can you purify more proteins if you increase the number of separation steps to two, and if so how?
E.g. by passing the mixture through first a cation and then an anion exchange column at pH 5.
If you elute the proteins by changing the pH, in what direction should the change be for cation and anion
exchange respectively?
Increase pH for cation exchange and decrease for anion exchange.
Are there other ways to elute the protein?
By increasing the ionic strength.
Homepage: http://aula.au.dk/courses/AULA100407093252/
Page 3/3