Analysis of SDS-PAGE
The interpretation of an SDS gel is usually straightforward.
The investigator looks for a particular band or
pattern of bands that are characteristic of the
biological sample that was collected. The gel is
examined to determine purity of the preparation
or the presence/absence/modification of specific
protein bands. SDS-PAGE can be combined with specialized
techniques such as immunoblotting, two-dimensional
electrophoresis, peptide mapping, etc. in order
to identify specific proteins or protein isoforms (two
or more versions of the same protein with slightly
An objective of this part of the study is to
learn some of the strategies for recognizing protein
bands and patterns on a gel. Another objective
is to learn the limitations of the technique -
identification of a band on a protein gel is not
considered positive proof of identity. One more
objective is to learn to improve one's performance
by recognizing the consequences of imperfect technique,
and the disasterous consequences of major errors
in gel/sample preparation and running gels.
Image the gel
We used to have to take photographs of gels for
analysis, scan them on densitometers, or, if we
were really desparate, photocopy the gel directly.
Now that high quality digital images can be routinely
made, scanning and digitizing a gel image is the
most convenient way of preparing an image for analysis.
An important original gel should be dried down
Critique your gels
Examine each of the gels on which your samples
have been run, and using the poster materials (see
the guide) evaluate your performance. If something
went wrong, the "hall
of shame" should help you identify the
Identify gels, protein lanes, and obtain relative
As silly as it sounds, it is important to identify
which gel is which (percent acrylamide and which
samples were loaded), top and bottom of each gel,
and the lanes corresponding to specific samples.
All of that information, including identifying
marks and labels, should be recorded in the notebook.
The top of the gel is the top of the separating
gel. The stacker is of no use now and can be removed.
If the dye front is not visible, either it ran
off the gel or is not visible because no proteins
ran with the tracking dye (usually with high percent
acrylamide gels). Each band of importance should
be labeled (without obscuring information on the
image) and a relative mobility determined for each,
using either the dye front or, if the gel has calibrated
standards, the bottom of the gel as a reference.
Relative mobility is determined by measuring the
distance from the top of the gel to the middle
of the dye front or arbitrary reference point,
measuring the distance from the top of the gel
to the middle of the band, and dividing the second
measurement from the first. This is the Rf, which
is always between 0 and 1.
Note that the relative mobility of a given protein depends
on gel concentration. Any single gel has
an upper and lower limit to its useful range
for estimating molecular weights.
The relationship between relative mobility and
molecular weight is logarithmic, in a perfect gel
of uniform concentration. Your results will always
be a bit less than perfect, however a standard
curve will turn out nearly linear when a log scale
is used. On semilog paper, or using a graphics
program with y-axis on a log scale, the molecular
weight of each calibrated standard should be plotted
on the y-axis, versus its Rf on the x-axis.
Obtain apparent molecular weights
The relative mobility of a polypeptide in SDS-PAGE
is typically related directly to the log of its
molecular weight. However many factors act to modify
the migration of individual polypeptides, so that
the molecular weight determined from a gel is seldom
identical to the molecular weight that would be
obtained from the actual amino acid sequence. Nonprotein
substituents such as carbohydrate or lipid residues
can exercise 'drag' on the polypeptide. Denaturation
may be incomplete for polypeptides with long stretches
of hydrophobic residues, which is true of many
transmembrane proteins. A protein may have a large
proportion of acidic or basic residues, which alter
its charge-to-mass ratio. Despite one's best efforts,
the protein may suffer some degradation, leading
to a faster rate of migration than for the intact
The molecular weight as determined from SDS-PAGE
is called an apparent molecular weight.
Often it is close to the true molecular weight
of the polypeptide, but sometimes the apparent
molecular weight can be way off - as is the case
for one or more erythrocyte membrane proteins.
By the way, molecular weight and molecular mass
are quantitatively identical, but molecular weight
is a relative term thus the quantity is dimensionless.
The position of each unknown band relative to
the protein standards should be noted before using
the standard curve. The standards may not fall
exactly on the curve fit even though they were
specifically chosen because their apparent molecular
weights are very close to their true molecular
weights. A curve fit with a straight line may not
intersect the topmost or bottommost standard. Because
the relationship is logarithmic, an error at the
bottom is no big deal. However an error at the
top of the gel can result in a huge over- or underestimation.
It might be a good idea just to connect the data
points and interpolate apparent molecular weight
directly, ignoring the 'curve.'
The molecular weight that corresponds to the
Rf of the polypeptide band is read from the standard,
and recorded with a reasonable number of significant
figures. Precision can be estimated from the minimum
resolution on a gel (usually ~0.5 mm) and the molecular
weight range covered by that distance at that position
on the gel (each gel is more accurate toward the
bottom than toward the top).
Characterize the membrane-associated
See the topic "identifying
bands on protein gels" for a general
approach to characterizing the protein composition
of a fraction.
SDS-PAGE of purified organelles such as plasma
membranes, ribosomes, endoplasmic reticulum, etc.
usually gives patterns that are dominated by one
or a few major protein bands. On a 7 to 8% gel,
erythrocyte membranes yield a pattern that is dominated
by: the heavy (>200 kd) spectrin bands, which form
a doublet at the top; the wide anion exchanger
protein band (band 3) in the range of 100 kd or
so; and the actin band at around 43 kd. An investigator
should look for the pattern, double check that
the apparent molecular weights are in the right
ball park, and use the basic pattern as a framework
for identification of less dense bands.
If the pattern is not apparent, there may have
been a problem with the fractionation, sample preparation,
or the gel. The results should be compared with
illustrations showing electrophoresis of red cell
membrane preparations, and the procedures double
checked if the pattern isn't at least somewhat
close to the typical pattern. Degradation of proteins
or failure of the reducing agent are common reasons
for such 'strange' results.
The current model for
the structure of the red blood cell membrane should
be used as a guide to identification, along with
other sources of information. The molecular masses
of the known proteins have been established. The
number corresponding to molecular mass is identical
to that corresponding to molecular weight (e.g.,
molecular mass of 200 kDa corresponds to molecular
weight of 200,000). Apparent molecular weights
are only one basis for identification, and should
not be relied upon solely. Also consider quantity
of protein (indicated by intensity of a band),
the fraction(s) in which a band is found, associations
with other bands, and quality of a band. As tentative
identifications are made, one must consider the
uncertainty inherent in the technique. Some proteins
may not show up at all, because they are present
in too few numbers or they don't stain with the
As mentioned previously, 'nonprotein' residues
on polypeptide chains can influence migration,
leading to indistinct bands and deviation of apparent
molecular weight from the true molecular weight.
Proteins with a high carbohydrate content are notorious
for migrating with unpredictable relative mobilities,
and for failing to stain with standard methods.
Species differences and differences in method of
determination of published molecular weights can
also lead to disparities between an estimate and