Introduction
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) is a reliable method for determining the molecular
weight (MW) of an unknown protein. The first step in MW
determination of a protein is to separate the protein sample
on the same gel with a set of MW standards. Next, a graph
of log MW vs. relative migration distance (Rf) is plotted, based
on the values obtained for the bands in the MW standard.
The MW of the unknown protein band is then calculated by
interpolation using this graph. The key to determining MW
accurately is selecting separation conditions that will produce
a linear relationship between log MW and migration within the
likely MW range of the unknown protein.
SDS-PAGE Conditions for MW Determination
To ensure accurate MW determination, both the protein
standards and the unknown protein must be electrophoresed
on the same gel under identical separation conditions. It is also
important to generate multiple data points (at least three gels)
so that the estimated MW has statistical significance. The
sample buffer used to solubilize the proteins should contain
reducing agents (dithiothreitol or β-mercaptoethanol) to break
disulfide bonds, which minimizes the effect of secondary
structure on migration. In addition, a strong ionic detergent
such as SDS is a required component of the sample buffer.
SDS provides two functions: It denatures secondary, tertiary,
and quaternary structures by binding to hydrophobic protein
regions, and its binding confers a net negative charge on the
proteins, which also results in a constant charge-to-mass ratio.
The proteins are then separated through a gel in an electrical
field according to their mass. However, other factors may also
influence protein separation. These factors are discussed
briefly under Limitations.
Analysis of Electrophoresed Proteins
An example of the approach is shown in Figure 1 using Green
Fluorescent Protein (GFP) as a hypothetical example of an
unknown protein. A dilution series of an E. coli lysate spiked
with GFP and Precision Plus Protein™unstained standards
was electrophoresed in different lanes of a Criterion™4–20%
SDS-PAGE gel, then stained with Bio-Safe™Coomassie stain
and destained in distilled water to visualize the protein bands.
The gel was then analyzed to obtain the Rfvalues for each
band. The Rfis defined as the migration distance of the protein
through the gel divided by the migration distance of the dye
front. The distance should be measured from the top of the
resolving gel to the band of interest, as illustrated on the gel.
A plot of log MW versus Rf(Figure 2) was generated from the
bands in the gel shown in Figure 1 to determine the MW of
the unknown protein. The simplest method for this is to base
the MW determination on a standard curve. If the curve is
nearly linear, it can be described by the formula y = mx + b,
where y is the log MW, m is the slope, x is the Rf, and b is
the y-intercept, as shown in Figure 2.
Alternatively, Bio-Rad’s The Discovery Series™Quantity One®
1-D analysis software, or software with similar capabilities, can
be used to determine the Rfvalues. Quantity One is a tool for
imaging and analyzing 1-D electrophoresis gels, dot blots, slot
blots, and colony counts. For accurate MW determination,
the unknown protein should be within the linear range of the
standard curve, and the amount of the unknown protein
(or its intensity after staining) should match the corresponding
electrophoresis tech note 3133
Molecular Weight Determination by SDS-PAGE
1234567
250
150
100
75
50
37
25
20
15
10
Top of resolving gel
Dye front
Unknown band
Migration
distance
of dye front
(67 mm)
Migration
distance of
unknown
band
(45 mm)
Fig. 1. Example showing approach for MW determination of an unknown
protein. Lane 1, 10 µl of Precision Plus Protein unstained standards; lanes 2–7,
a dilution series of an E. coli lysate containing a hypothetically unknown protein
(GFP). Proteins were separated by SDS-PAGE in a Criterion 4–20% Tris-HCl
gel and stained with Bio-Safe Coomassie stain. Gel is shown actual size.
MW standards are in kD.
