Calculate rf value gel electrophoresis

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.