CHEM 3380 Sp16 Quiz 3

Name______KEY____________

Use correct significant figures in your final answer if appropriate.

Electrophoretic Methods:
1. (2 pts) On the following picture of a gel show where the cathode would be
located relative to the gel when it was running (using a minus sign, -) and
where the anode would be located (using a + sign).
Cathode (-)

direction of migration
Anode (+)
2. (2 pts) Assume that the gel shown in question #1 was run as an SDS-PAGE
gel under reducing conditions. Also assume that a protein homodimer was one
of the proteins resolved by the gel. (The homodimer has two subunits that are
equivalent in amino acid sequence and are connected with a disulfide linkage in
the native form.) The molecular weight of the native protein (as a dimer) is 50
kDa. What apparent molecular weight would you expect to see for migration of
the protein under the SDS-PAGE, reducing conditions?
25 kDa under reducing conditions, the disulfide linkage will be reduced and
each subunit will migrate independently.

3. (2 pts) Consider the following gel and

Western blot shown below the gel. Briefly
explain what steps are required to go from the
SDS-PAGE gel at top of the picture to the
Western blot at the bottom showing Nicastrin
and PS1-NTF only.
1. blot proteins from gel to membrane
2. expose membrane to primary antibody for
Nicastrin
or
PS1-NTF
(after
blocking
nonspecific sites on membrane)
3. expose membrane to secondary antibody
and visualize based on type of secondary

4. (2 pts). Draw a gel to show nondenaturing electrophoresis of the following

mixture of proteins:
lysozyme (13.9 kDa) actually pI of lysozyme is high, so gel would probably not
look as well-resolved as what is drawn below
myoglobin (15 kDa)
hemoglobin 4 subunits (63 kDa)
Make sure you include a molecular weight marker for the gel.

63 kDa

15 kDa

5. (2 pts) Agarose gel electrophoresis is commonly used to separate supercoiled

plasmid DNA from linearized DNA. Why is it possible to use agarose gel
electrophoresis to separate supercoiled plasmid from linearized plasmid DNA
for the same plasmid when the two have the same molecular weight?

Agarose gel electrophoresis is non-denaturing, so shape, charge, and

molecular weight influence migration of biomolecule in the gel. Supercoiled
plasmid DNA is more compact than the same sized linearized plasmid, so the
supercoiled form will migrate further in the gel.