Answers to exercises in electrophoresis

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.

The mobility increase with Z and with 1/R0

Deamidation (from Asn to Asp, or Gln to Glu) is a post-translational modification that adds a negative
charge to a protein without changing the mass significantly. What type of gel would be useful to detect
deamidation? Which way should the current go if the protein is negatively charged in the buffer system
chosen? Describe how the unaltered and deamidated proteins would migrate.
A native gel. Migration from to +. Deamidated migrates faster.
What is the purpose of a stacking gel, and how does it work?
Stacking gels give sharper bands and allow larger samples to be loaded.
The system utilizes a stacking gel of pH 6.7 with a low concentration of acrylamide and resolving gel of
pH 8.9 and a higher concentration of acrylamide.
Glycine is incorporated in the reservoir buffer. At pH 6.7 glycine is close to neutral, while it is negatively
charged at pH 8.9. A lower ionic strength increases the electric field.
The increased electric field together with the weaker matrix increases the mobility of the protein in the
stacking gel compared to the resolving gel.
Protein which has to travel a longer distance in the stacking gel will catch up with the rest of the protein
when reaching the gel boundary. Cf. traffic jam.
What does SDS do to the protein is SDS page?

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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.

Briefly describe four (surprise) methods for liquid chromatographic separation.

Affinity (biospecific) chromatography separation by specific binding
Ion exchange chromatography separation by charge
Reverse phase high performance liquid chromatography separation by hydrophobicity
Gel filtration chromatography separation by size
Are there any limits for which buffers and columns that may be used for a certain protein?
The protein must be soluble in the buffer and the column material must be stable in the buffer.

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?

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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.

5. Reversed phase HPLC

a Which of the solvent and the stationary phase is the most hydrophobic in RP-HPLC?
The stationary phase is most hydrophobic.
b What characteristic of the molecule the basis for separation by RP-HPLC?
The partition coefficient between solvents of different polarity.
c Why does a longer column often give better separation? (You might want to discuss the effects by
comparison with separation funnels and distillation columns.)
In a separation funnel an equilibrium between the two phases are established. In a column there is a
number of coupled equilibria similar to the plates in a distillation column.
d Are there effects that make the separation worse with a longer column?
The longer the column the more random diffusion. Higher pressures will also be needed.
e Give four examples of factors that you might change to control the selectivity of the separation.
Mobile-phase solvent, mobile-phase pH, stationary phase, temperature.
f
What is gradient elution and why might one want to use it?
The solvent conditions are gradually changed. Peaks are generally sharper which result in increased
sensitivity of detection and more concentrated solutions.
6. Gel filtration Size exclusion chromatography
a Describe how gel filtration works: Resin, basis for separation etc.
Macromolecules are separated based on their different ability to penetrate into pores of molecular
dimensions in a matrix. As small molecules penetrate deeper into the pores than large proteins, they will
be retarded and elute later than large proteins.
b When gel filtration is used in protein purification, it is usually used in one of the latter steps. Any idea
why?
Gel filtration columns are generally expensive and the pores will easily get clogged if a dirty sample is
put on.
c Does the degree of folding affect the rate of elution in a gel filtration column, and if so how?
An unfolded protein has a larger hydrodynamic radius and will therefore elute slower.
7. Protein purification
Invent two proteins with certain characteristics and design purification processes for them.
Good luck!

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