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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