Electrophoresis and related techniques
1.
Tanya was attempting to determine the molecular weight of the milk protein βlactoglobulin. SDS-PAGE of the purified protein gave a single band with a molecular weight of
18 kDa. She ran a gel filtration column, loading a 1 mg sample of protein in a volume of
100 µL. The elution volume led her to predict a molecular weight of 36 kDa. When she
repeated the procedure (as all good biochemists should), she was running low on pure protein,
and loaded a 10 µg sample in 100 µL. This time, the elution volume indicated a molecular
weight of 25 kDa. Realizing that something odd was happening, she ran samples of protein
containing 500 µg and 100 µg in 100 µL, and obtained molecular weights of 35 kDa and 32 kDa
respectively. Can you explain the data?
The protein is a dimer, and dissociates (rapid, reversible equilibrium) at lower
concentrations.
2.
A protein mixture separated by gel filtration chromatography contains proteins Mr 120150 kDa. When analyzed by non-denaturing gel electrophoresis, bands appear
throughout the gel. When analyzed by SDS-gel electrophoresis, there are some bands
between 120 and 150 kDa, but also several bands at 75 kDa or less. Why might the three
techniques give different results?
Gel filtration separates on the basis of size (and shape, to some extent). Therefore, a
group of fractions taken at a certain range of elution volumes will have a limited range of sizes
(120-150 kDa in this case). In the non-denaturing gel, separation is on the basis of charge, size,
and shape. A protein with low mobility may have low charge or be large or highly elongated. A
protein at the bottom of the gel (high mobility) could also be large, but spherical and highly
charged. No simple prediction of properties can be made from the gel. [MW may be estimated
from a plot of mobility vs. gel % (Ferguson plot); see, e.g., Figure 2 in Maeda et al., J. Biol.
Chem. 276: 29833-29838, 2001.]
On the SDS gel, proteins are denatured. Size alone determines separation, due to the
functional effect of the gel. Bands at 120 to 150 kDa represent proteins formed from a single
polypeptide chain. However, because of denaturation, proteins that are dimers, trimers and
tetramers, etc. fall apart, and the constituent polypeptide chains migrate separately in SDSPAGE. Bands of 60-75 kDa represent proteins that are dimers; bands of 40-50 kDa might be
trimers (relatively uncommon) and 30-37 kDa would be tetramers (quite common). Conversely,
if a protein gives a band of 30 kDa in SDS-PAGE, in the absence of other knowledge, you can't
assume that it will be 30 kDa in gel filtration as well.
3.
Plot a calibration curve for the Fermentas™ pre-stained MW markers (the gel is shown in
the course manual) by measuring the (relative) distance migrated by each band and
plotting this versus log(MWr), using the data given in the list of proteins.
Fermentas standards
175
150
migration
(mm)
125
100
75
50
lys
lact
ca
ov
bsa
gal
25
0
10
100
MW (kDa)
4.
A tetrameric enzyme consists of two α subunits (25 kDa) and two β subunits (40 kDa).
The α subunits are joined to each other by -S-S- bridges, as are the β subunits. Indicate
the SDS-PAGE pattern that you would expect to see in the absence and in the presence
of β-mercaptoethanol.
In the presence of β-mercaptoethanol, all of the -S-S- bridges are broken, and we would
see two bands on the gel corresponding to the two subunits, at molecular weights of 25 kDa and
40 kDa, respectively.
In the absence of β-mercaptoethanol, the -S-S- bridges will remain intact, so that the
enzyme will be dissociated into α2 and β2 units. We would thus see two bands on the gel at
50 kDa and 80 kDa, respectively.