Biol 340, Cell Biology
Collecting Raw Data from your Sheep RBC Plasma Membrane Protein Gel
Data Collection: An Overview
The objective of the type of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) we ran in lab is
to determine the molecular weights of the sheep RBC plasma membrane protein bands resolved on your
gel. In SDS-PAGE, the molecular weight of a protein is inversely related to the distance the protein
migrated through the gel. That is, the smaller the protein, the faster it migrates through the gel. Thus, the
determination of protein molecular weight in SDS-PAGE is based on measuring the distance of migration of
each protein band.
In electrophoresis, migration distance is expressed in terms of relative mobility (Rf). As defined in
the paper chromatography experiment early in the semester, relative mobility is the ratio of the distance the
protein migrated (dsample) to the distance the solvent front migrated from the origin (d solvent) of migration
(see equation 1).
Rf = dsample / dsolvent
(1)
So, the raw data you will collect from your gel will be dsample and dsolvent data. Once the raw data are
collected, the relative mobility of each band is calculated.
Inspecting Your Stained Gel
You instructor will make a photograph of your gel available to you. Your gel probably looks like the
one shown in figure 1 below. If you remember, a set of protein standards was loaded into the first two
wells, and you followed, loading your mixture of RBC plasma membrane proteins into two other wells.
Figure 1 identifies the six standard proteins in lane 1 and their molecular weights. The figure also labels
the major bands in your mixture from Bands 1a and 1b at the top to Band 8 at the bottom. Below Band 8 is
the bromophenol blue line that marks the solvent front.
Figure 1. Identification of the standards and the major RBC plasma membrane protein bands.
Lane 1 (far left) contains the six standard proteins. Lanes 2 - 7 contain mammalian RBC plasm
membrane samples.
If you haven’t done so yet, pull up the photograph of your gel on your computer monitor and look it
over. The photograph of your gel is better viewed on a computer monitor rather than on a printout. The
six standard protein bands are clearly visible in the photograph of your gel. The visibility of the RBC
plasma membrane protein bands, however, will likely vary from band to band. Bands 1a and 1 b should be
clearly visible. Bands 2.1 and 2.2 probably appear as one thin band, lets simply call it Band 2. Band 3
should appear as a wide band. Band 4.1a and 4.1b will appear as two bands below Band 3, and Band 4.2
will appear below that, lined up with the 4tth standard protein band (Serum Albumin). Band 5 will be lined up
with 5th standard protein (Ovalbumin). Bands 6, 7 and 8 might be very faint - we are going to ignore them.
The Standards and the Standard Curve
The standards are known proteins of known molecular weights. Since we know their molecular
weights and since we can determine their relative mobilities from the gel, we use these standards to
determine the precise mathematical relationship between relative mobility and molecular weight for your
gel. Since the relationship between molecular weight and migration distance can be different for each
gel, a set of standards must be run on every gel, and one must use the standards run on one’s own gel. The
standards run on someone else’s gel are not valid for your gel.
The standard mixture we used in lab is a commercially available mixture containing the proteins listed
in the table below.
Protein standard
Molecular Weight (daltons)
myosin
200,000
beta galactosidase
116,250
phosphorylase B
97,400
bovine serum albumin
66,200
egg albumin
45,000
carbonic anhydrase
31,000
Before you can determine the molecular weights of your RBC plasma membrane proteins, you must collect
the migration data for each of the six standard protein bands and calculate their respective relative mobilities.
Later, you will need to graph the molecular weight of the standards vs. relative mobility. This curve is called a
standard curve.
Standard curves can be plotted in several ways. The two most-basic ways are shown in figures 2a and
2b. When molecular weight is plotted against relative mobility, as is done in figure 2a, a curvilinear plot is
produced. However, when the log of the molecular weight is plotted against relative mobility, as is done in figure
2b, a straight-line plot is produced.
Figure 2a. A standard curve of the
six standard proteins identified
above. Molecular weight is plotted
against relative mobility, generating
a curved line. This is only an
example. You will be producing
your own.
Figure 2b. A standard curve of the
six standard proteins identified
above. In this case, the log of
molecular weight is plotted against
relative mobility, generating a
straight line. This type of standard
curve, one that yields a straight
line, is preferred to that shown in
fig 2a.
Generally, the precision of a straight-line is greater than the precision of a curved line, so the type of
standard curve shown in figure 2b is preferred. Note that in this type of graph, the straight line does not
connect the data points, but rather is drawn among the data points. This straight line represents a best-fit
line that can only be fitted to the data by computer. Microsoft Excel can perform this type of graph easily,
and it can provide the equation of the line. The equation of the line is important. The user can plug the
relative mobility of any one of the RBC plasma membrane protein bands into the equation and calculate the
log of the molecular weight and the molecular weight of that protein band. This is precisely what you will be
doing in your analysis with Microsoft Excel.
Collecting The Raw Data
1. First, collect the raw data for the standards.
Inspect the two standard lanes (lanes 1 and 2) on your gel for reproducibility. If the reproducibility is
satisfactory, use the middle most standard lane, lane 2 (it's probably the most reliable), and measure the
migration distance (D sample) in mm (whole numbers only please) from the bottom of the well (the origin) to
the center of each standard protein band. As you collect the data, record the data below.
Now measure the migration distance of the bromophenol blue line (D solvent) in mm (whole number only)
from the bottom of that same well. Record this number in each of the six cells below.
Protein Standard
myosin
beta galactosidase
phosphorylase B
bovine serum albumin
egg albumin
carbonic anhydrase
D Sample
(mm)
D Solvent
(mm)
Rf
Molecular
Weight
200,000
116,250
97,400
66,200
45,000
31,000
Log10
M.W.
2. Next, collect the raw data for your RBC plasma membrane protein bands
Collect and record the raw data for your RBC plasma membrane protein bands in the same way you did for
the protein standards. Inspect your two sample lanes for reproducibility. Assuming the lanes look alike,
pick one of them for your data, and measure the migration distance of each protein band (D sample) in mm
(whole numbers only please) from the bottom of the well (the origin) to the center of each band. As you
collect the data, record the data below.
Now measure the migration distance of the bromophenol blue line (D solvent) in mm (whole number only)
from the bottom of that same well. Record this number in each of the nine cells below.
RBC Plasma
Membrane Protein
Band
D Sample
(mm)
D Solvent
(mm)
Rf
Molecular
Weight
Log10
M.W.
band 1a
band 1b
band 2
band 3
band 4.1a
band 4.1b
band 4.2
band 5
band 7
Once you have collected all the raw data, you are ready to perform the calculations needed to create your
standard curve and determine the molecular weights of your unknowns. Your course instructor will provide
additional instructions.
Copyright © 2011 Stephen Gallik.