Lab 8, Day 3: SDS-PAGE data analysis
Objectives
After completing this lab, you should be able to:
•
•
•
•
Calculate the Rf values of proteins from the photograph of a stained SDS-PAGE gel
Plot a protein marker standard curve
Use the standard curve to calculate the molecular weights of native and denatured GFP of BFP
Evaluate the effectiveness of protein purification via column chromatography from gel electrophoresis
results
Important note: There is NO Prelab for this lab!
Lab Procedures
To complete the Lab Procedures, you will need a metric ruler and a copy of your group’s polyacrylamide
gel picture (page 6).
Recall the recommended order of the protein samples and their contents, from left to right:
Lane 1: G Nat = Purified GFP in its native state
Lane 2: G Boil = Purified and denatured GFP
Lane 3: IG Boil = Protein mixture (GFP and other proteins) that was denatured
Lane 4: Protein marker
Lane 5: B Nat = Purified BFP in its native state
Lane 6: B Boil = Purified and denatured BFP
Lane 7: IG Boil = Protein mixture (BFP and other proteins) that was denatured
I. Measuring protein marker band migration distances
1.
In your gel picture, locate the lane that contains the protein marker. If you followed the
recommended loading order, this would be Lane 4. The blue band that traveled the furthest from the
well represents the tracking dye (see picture on next page). This tracking dye band should be visible
in all lanes with denatured protein samples: G Boil, B Boil, IG Boil, IB Boil. It will be absent in the G
Nat and B Nat lanes.
Don’t have a ruler at home? Use the online ruler here. Now measure from the bottom edge of the
well to the tracking dye band that is in the same lane (directly below this well). This is the migration
distance of the tracking dye. Note the units (mm or cm) you use to measure this migration distance.
Tracking dye migration distance from well =
ACC BIOL 1406 Lab Manual Cypress Creek Edition
Lab 8, Day 3
____________
Page 1
2.
The picture below shows the general pattern of the seven protein marker bands that you might see in
the lane, along with the protein molecular weights in Daltons (Da) that they represent.
Use this picture to help you identify each band that appears in your gel’s protein marker. Because
each gel is different, you may not see all seven bands. Examples: The 6000 Da band is often
faint and hard to see. The 4000 Da band might travel as far as the tracking dye, so you might
not be able to distinguish them from each other.
3.
In your gel picture, use a ruler to measure the migration distance from the bottom edge of the well to
each marker band. Use the same metric units as the tracking dye migration distance. For
consistency, measure to the same point in each marker band (top edge, bottom edge, middle of
band).
Record the distances in the table below. Leave the migration distance blank if the band is absent
from your gel picture.
Protein marker band
migration distance from well
(Units = ___________ )
Protein marker
molecular weight
(Da)
Bottom edge of well
98,000
98,000 Da
64,000
64,000 Da
50,000
50,000 Da
36,000
36,000 Da
15,000
6,000
15,000 Da
4,000
6,000 Da (often faint or not visible)
4,000 Da
)
(Tracking dye migration is the same
or just below this marker)
II. Creating a protein marker standard curve
You have already drawn standard curves for other lab exercises. These graphs helped you to calculate
unknown concentrations from known absorbances. The standard curve that you will draw for this lab will
help you to calculate the unknown molecular weight of a protein from a known R f.
To create this standard curve, you will graph the Rf of each marker band you identified in your gel (X) vs.
the log of the protein’s molecular weight (Y). Thus, your finished standard curve will not be shaped like a
curve at all - expressing marker migration as Rf and converting protein molecular weights to their logs will
result in a linear arrangement of points when the graph is drawn.
ACC BIOL 1406 Lab Manual Cypress Creek Edition
Lab 8, Day 3
Page 2
1.
For each band in your protein marker lane, use a calculator to:
a.
Calculate the Rf using the migration distances you measured in Part I:
Rf =
b.
Protein marker band migration distance from well
Tracking dye migration distance from well
Calculate the log of the protein marker molecular weight. The molecular weights are
given in the previous table. Enter the molecular weight into the calculator and press the “log”
button (or the other way around). The logs you calculate will be values smaller than 5.0.
Record the Rf and log values in the table below. If your gel had less than seven marker bands, you
might not use all of the rows in this table. These are unitless values.
Protein marker band
Rf
2.
Log of protein marker
molecular weight
Create the protein marker standard curve:
a. Using a computer program like Excel, create a scatter plot of protein marker band R f (X-axis) vs.
log of protein marker molecular weight (Y-axis).
b. Draw a linear regression trendline for your scatter plot. Show the equation for the trendline on the
graph.
If you have plotted your points correctly, the trendline will have a negative slope, which means
that protein marker migration distance and molecular weight are inversely related.
Format your graph (title, axes, etc.) according to the guidelines specified by your
instructor. You will turn in a copy of this protein marker standard curve for Postlab
Question 2.
ACC BIOL 1406 Lab Manual Cypress Creek Edition
Lab 8, Day 3
Page 3
III. Calculating Rf values for native and denatured GFP and BFP
1.
Transfer the tracking dye migration distance from Page 1:
Tracking dye migration distance from well = __________
2.
Locate the “G Nat” and “B Nat” lanes in your gel picture. In these two lanes, identify the protein band
that corresponds to native GFP or BFP. If your column did a good job of purifying these proteins, the
GFP or BFP will be the only band in the lane. If multiple bands are present, the native GFP or BFP
should be the darkest one.
Measure the migration distances of the native GFP band in G Nat and the native BFP band in B Nat,
again starting from the bottom edge of the well. Remember native GFP and BFP are dimers.
Native GFP migration distance from well = __________
Native BFP migration distance from well = __________
3.
Locate the “G Boil” and “B Boil” lanes in your gel picture. In these two lanes, identify the protein
band that corresponds to denatured GFP or BFP. Again, if your column did a good job of purifying
these proteins, they will appear as the only band in the lane. If multiple bands are present, the
denatured GFP or BFP should be the darkest one.
If the “G Boil” or “B Boil” lanes have no bands at all, use darkest band that appears in “IG Boil” or “IB
Boil”.
Measure the migration distances of the GFP band in G Boil (or IG Boil) and the BFP band in B Boil
(or IB Boil), again starting from the bottom edge of the well. Remember that denatured GFP and
BFP lack all levels of protein structure except for primary structure.
Denatured GFP migration distance from well = __________
Denatured BFP migration distance from well = __________
4.
Divide the four migration distances from above by the tracking dye migration distance to calculate R f
values. Transfer these calculations and answers to complete Postlab Question 3.
Native GFP Rf = _____________
Denatured GFP Rf = _____________
Native BFP Rf = _____________
Denatured BFP Rf = _____________
To answer Postlab Question 4, you will plug these R f values into the trendline equation from the
protein marker standard curve to calculate the molecular weight of GFP and BFP in their native and
denatured forms.
ACC BIOL 1406 Lab Manual Cypress Creek Edition
Lab 8, Day 3
Page 4
POSTLAB
1. In your gel picture, locate the lanes for IG Boil and for G Boil. IG Boil is a mixture of GFP and other
proteins (basically, GFP before purification by size exclusion chromatography). G Boil contains GFP
after purification by size exclusion chromatography.
How well did the size exclusion column (from Day 1) purify GFP from the rest of the proteins in the
protein mixture? You can evaluate this by first examining the appearance of the IG Boil lane and then
comparing its appearance to the G Boil lane. Compare these lanes according to the number of
different proteins that are present and intensities of the bands, especially of the band representing
GFP.
2. Include a copy of the protein marker standard curve you drew as part of the Lab Procedures. Be sure
that the graph is properly formatted: There is a descriptive title, the axes are clearly labeled, the data
points are shown as a scatter plot, and the linear regression trendline and its equation are also
displayed. Include any other graph formatting features required by your instructor.
3. Calculate for the Rf values of the following: Native GFP, native BFP, denatured GFP, and denatured
BFP. Show each calculation for full credit.
4. Use the equation for the standard curve trendline and the Rf values from Question 3 to calculate the
molecular weight of native GFP, native BFP, denatured GFP, and denatured BFP:
•
For each of these four calculations, plug the Rf into the trendline equation (where will you plug it
in?) and solve (which variable in the equation are you solving for?)
•
The answer you calculate (will be a small number) is not the protein’s molecular weight
yet! It’s the log of the protein’s molecular weight.
To fully solve for the molecular weight, use a calculator to take the antilog of your answer, which
basically undoes the logarithm. For many calculators, you would set up the antilog by pressing
the “Shift” or “2nd” button (or something similar) and then the “log” button.
The molecular weights you calculate should be relatively large values (in the thousands). The
units of molecular weight are Daltons. Show the calculations of each molecular weight for full
credit.
5. Is the calculated molecular weight of native GFP the same as denatured GFP? If the native and
denatured proteins differ in molecular weight, explain the reason for this difference.
6. The primary structures of GFP and BFP monomer proteins each contain 238 amino acids. However,
their primary structures differ in the identities of two of these amino acids.
Should the monomer GFP and BFP molecular weights be the same or different? Does your data
support your answer? (Compare the molecular weights you calculated for denatured GFP and BFP.)
ACC BIOL 1406 Lab Manual Cypress Creek Edition
Lab 8, Day 3
Page 5
SDS-PAGE
ACC BIOL 1406 Lab Manual Cypress Creek Edition
Lab 8, Day 3
Page 6
Lab 8_Day 3
Why?
Learn how to
• Calculate the Rf values of proteins on a SDS-PAGE gel.
• Plot a protein marker standard curve.
• Use the standard curve to calculate the molecular weights of native and
denatured GFP of BFP.
• Evaluate the effectiveness of protein purification via column chromatography
from gel electrophoresis results.
Lab 8_Day 3, you’ll analyze your gel results.
• Compare the difference in electrophoresis of the protein mixtures to the purified
GFP and BFP, to evaluate how well your column purified GFP and BFP from the
original protein mixtures.
• Compare the differences in electrophoresis of native GFP and BFP (dimers) to
denatured GFP and BFP (no longer dimers and without 3D shapes).
• Calculate the molecular weights of denatured GFP and BFP.
You will need a metric ruler and a copy of your group’s polyacrylamide gel
picture.
The order of the protein samples and their contents are listed below:
Lane 1: G Nat = Purified GFP that was not denatured
Lane 2: G Boil = Purified and denatured GFP
Lane 3: IG Boil = Protein mixture (GFP and other proteins) that was denatured
Lane 4: Protein marker
Lane 5: B Nat = Purified BFP that was not denatured
Lane 6: B Boil = Purified and denatured BFP
Lane 7: IG Boil = Protein mixture (BFP and other proteins) that was denatured
1 = G Nat
2 = G Boil
3 = IG Boil
M = Protein marker
4 = B Nat
5 = B Boil
6 = IB Boil
Tracking dye
1
2
3
M
4
5
6
Measuring protein marker band migration distances
In your gel picture (page 6), locate a lane that has the blue band that represents the
tracking dye.
The tracking dye band should be visible in the protein samples that protein
denaturing solution was added: G Boil, B Boil, IG Boil, IB Boil - it is the band that
migrated the farthest from the well and to the same point in all four of these lanes. It
will be absent in the G Nat and B Nat lanes.
Use a metric ruler to measure from the bottom edge of a well to the tracking dye
band that is in the same lane (directly below this well). This is the migration distance
of the tracking dye.
Note the units (mm or cm) you use to measure this migration distance.
Tracking dye migration distance = ____________
Protein Marker
Locate the lane in your gel that contains the protein marker.
There are seven marker bands that you might see in this lane with the protein
molecular weights in Daltons (Da):
98,000 Da, 64,000 Da, 50,000 Da, 36,000 Da, 15,000 Da, 6,000 Da ,4,000 Da
Because each gel is different, you may not see all seven bands. For example, the
6000 Da band is often faint and hard to see. The 4000 Da band might travel as far
as the tracking dye, so you might not be able to distinguish them from each other.
98,000 Da
64,000 Da
50,000 Da
36,000 Da
15,000 Da
4,000 Da
Calculate the relative migration (Rf) of each protein band in your gel.
Rf is the distance each protein band migrated through the gel relative to the
distance the tracking dye migrated through the gel.
To calculate the Rf for a protein band, use a metric ruler to measure (cm or mm)
two distances. The starting point for both measurements is the bottom of the well:
(1) The distance between the bottom of the well and the protein band. If your
protein bands differ in thickness, always measure this distance to the same point
of each band (for example, to the bottom edge of each one).
(2) The distance between the bottom of the well and the tracking dye
The Rf can then be calculated as follows:
Rf = (1) Distance from well bottom to protein band
(2) Distance from well bottom to tracking dye
Rf is a unitless value and will be a number that ranges from 0 to 1.
Calculate the relative migration (Rf) of each protein band in your gel.
http://www.ruf.rice.edu/~bioslabs/studies/sds-page/rf.html
Calculate the log of the protein marker molecular weight.
The molecular weights are given in a table on page 2 and
slide 6.
Enter the molecular weight into the calculator and press the
“log” button (or the other way around).
The logs you calculate will be values smaller than 5.0.
Convert data in table on
page 2 and record in
table on page 3.
i) Use migration
distance to calculate
Rf value
ii) Take log of MW
(value should be
between 0 – 5)
Example:
Log of MW 98,000
equals 4.99
Page 3
Protein marker band
Rf
Log of protein marker
molecular weight
4.99
Creating a protein marker standard curve (Graph)
You have already drawn standard curves for other lab exercises.
These graphs helped you to calculate unknown concentrations from
known absorbance's.
The standard curve you will draw for this lab will help you to calculate the
unknown molecular weight of a protein from a known Rf.
Create standard curve. You will graph the Rf of each marker band you
identified in your gel (X axis) vs. the log of the protein’s molecular
weight (Y axis).
Converting migration distances to Rf and converting the molecular weights
to the log of molecular weight will result in a linear arrangement of points
in the graph.
Create the protein marker standard curve:
Using a computer program like Excel, create a scatter plot of protein marker
band Rf (X-axis) vs. log of protein marker molecular weight (Y-axis). Be
sure to properly label the axes and provide a descriptive title at the top of
your graph.
Draw a linear regression trendline for your scatter plot. Show the
equation for the trendline on the graph.
If you have plotted your points correctly, the trendline will have a negative
slope, which means that the protein marker molecular weight and migration
distance are inversely related.
You will turn in a copy of this protein marker standard curve as part of
the Postlab.
Protein Marker Standard Curve
1 = G Nat
2 = G Boil
3 = IG Boil
4 = Protein marker
1
2
3
4
5
6
7
(Molecular weights in Lab 8-3)
5 = B Nat
6 = B Boil
7 = IB Boil
Tracking dye
•
•
•
Orange arrows indicate the GFP and BFP protein bands
White arrows indicate the protein marker bands (if your gel is missing a few, that’s fine)
Numbered markings on the ruler represent centimeters
Lab Report
1. Lab Procedures:
➢ Data and data in tables.
2. Postlab: Questions #1 - 6
➢ Insert protein marker standard curve (graph) for question #2.
➢ Also, add a labeled gel pic to the very end of the lab report.
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