Lab 6- Instructions for use of ImageJ for gel analysis

Biology 29 Cell Structure and Function Spring, 2009 Lab 6- Instructions for use of ImageJ for gel analysis I. Constructing a Standard Curve to determine Protein Molecular Weights A standard curve of molecular weights is made by plotting the relative migration distances (x-axis) of the proteins in a set of standards versus their known molecular weights (y-axis) on a semi-log graph. The relative distance (Rf) migrated by a particular protein is inversely proportional to the log10 of its molecular weight. By running a mixture of polypeptides of known molecular weights ("standards"), one can construct a standard curve showing this relationship. Then, by calculating the Rf of a particular protein on a gel, its molecular weight can be read off the standard curve. Some cautions: the apparent molecular weight you’ve determined from a gel can differ from the actual molecular weight. The estimated values for high molecular weight proteins will be less accurate than for smaller proteins because the scale of their migration is compacted; i.e., bands separate more distinctly the farther they travel down the gel. Some proteins are modified by phosphates or oligosaccharides, which cause them to migrate differently than expected. Finally, the standard curve is not strictly linear, even when plotted on semi-log graph paper. The log function, however, changes slowly enough that the straight line gives reasonable molecular weight estimates. A. Scanning and Obtaining a Digital Image of your Gel Your lab instructor will provide you with a digital image, in TIFF format, of your gel, obtained from a scanner. You can copy the file to your network space or to a flash drive so that you have it available to analyze. B. Analyzing Gels with Image J See Appendix I on page 5 if you need to download and install Image J. 1. Open the Image J program by double clicking on the microscope icon, and then open the digital image of your gel (File > Open). (If you need to reset the image at any time during the analysis, use File > Revert). 2. Your lanes must be horizontal in order for Image J to analyze them properly. You can rotate your image if needed using Image > Rotate. The top of the gel (where the wells are) should be on the left. The lane at the bottom of the image is then Lane 1. 3. To crop the image so that it includes just the gel, select the rectangular selections tool from the Image J toolbar. Draw a rectangle around the gel portion of the image. Image > Crop. Save this cropped image in case you have to re-open the file. 4. To obtain the right measurement parameters, open Analyze > Set measurements, and uncheck all boxes. Set decimal places to 1. 1 5. Analyze > Gels > Gel Analyzer Options. Make sure the “invert peaks” box is checked. 6. With the rectangular sections tool, first enclose the molecular weight standard lane (lane 4) on your gel with the rectangle box. Start at the far left edge of the image so that the X coordinate reads zero. Drag the cursor to the opposite corner of the lane, all the way over to the right edge of the image. Adjust the size of the rectangle so that it includes only bands from the lane of interest (the rectangle can be quite narrow, as long as it includes a part of each band in the lane). 7. With a rectangle enclosing the first lane, Analyze > Gels > Select First Lane. This will mark the first lane you have chosen as number 1. 8. Drag the rectangle (don’t draw a new one) to the next lane of interest (E.g. Lane 3, Whole cells), centering it so that the middle part of the bands in that lane fall within the rectangle. Then Analyze > Gels > Select Next Lane. Continue to do this for one of each of the duplicated samples. Each lane will be numbered in the order in which you selected them. 9. After all the lanes of interest have been selected, Analyze > Gels > Plot Lanes. This creates a series of line graphs relating the density of the color in each band to the distance migrated. Each lane is plotted separately. The first lane highlighted (in this case, Lane 4, the molecular weight standards) is the first plot (at the top), and the second lane is the second plot, etc. Note: to redo “Plot Lanes” it is necessary to start over at “Open File”, this is an unfortunate software glitch and you may need a few tries before you get everything 2 the way you want it. Be sure to save the cropped image of your gel (step 3) to make this easier. 10. Using the Scrolling tool from the Image J Toolbar (the hand), move the graphs up and down to see the graphs from all lanes you selected. 11. Move to the molecular weight standards graph (lane 4, the graph at the top). Select the Point Selections tool ( ) from the Image J Toolbar. Mark the desired points (the tips of the most intense peaks, one for each known molecular weight) with the Point Selections tool, as shown in the figure below (the dots show where the peaks have been marked). To check that you are marking the peaks that correspond with the bands, move back and forth between the image of the gel and the graph. Rest (don’t click) the cursor on the band of interest and note its X coordinate. Then rest the cursor at the tip of the peak and check its X coordinate. Their locations should be nearly the same (within a few pixels). Make sure that you have as many measurements as known molecular weight bands in this sample (10). Plot of band intensities for two lanes. The first graph has the first four major bands of the lane marked with the Point Selections tool 12. After marking the tips of the peaks, type Analyze > Measure, a table will appear in a new window. The table will include the point number, X, Y, Z and Value. X and Y are the current cursor coordinates, with the origin at the left-hand corner. We are concerned with only the X value. You will have to keep track of which measurements correspond to each lane (numbers in the left-hand column refer to the order in which you marked the tips) 3 13. For the other three bands, measure the four most prominent bands from the left (top of the gel). You will have to move back and forth between the Scrolling tool and the Point Selections tool to see and mark all of the graphs you will be measuring. Remember to keep track of which measurements correspond to each lane. 14. Make one more measurement. Click on the point that represents the very end of the gel (i.e. the point furthest away from the wells). This number represents the dye front and will be important in calculating the Rf values for each band. 15. Save the table as an Excel file (.xls) by using the menu above the table (not the Image J tool bar) (File > Save as). Open the Excel file and delete the Y, Z, and Value columns (save the X coordinates only). A suggested way to organize the data is shown below. Remember that the last row of data in the table represents the position of the dye front. Band 1 2 3 4 5 6 7 Label standard standard standard standard Lane 2, band 1 Lane 2, band 2 dye front X 462 518 584 616 477 601 657 Standard MW (kD) 100 70 40 15 Rf log MW 0.70 2 0.79 1.85 0.89 1.60 0.94 1.18 0.73 1.99 0.91 1.38 MW of unknowns 97.20 23.78 C. Constructing the Molecular Weight Standard Curve 1. Begin by determining the Rf values for all of the bands by dividing the X coordinate for each band by the X coordinate for the dye front (the last measure you took). (It is very convenient to do these calculations as part of the Excel file you just made. Ask your lab instructor for help if you don’t know how to enter equations into Excel). Decrease all calculations in the table to 2 decimal points. 2. Insert a column to the left of the Rf values and enter the known molecular weights of the molecular weight standard bands (the bands from lane 4; the first 10 measurements in your table). In the column to the right of the Rf column, calculate the log10 of the molecular weights, the equation is =log (cell#). 3. Using Excel, construct a standard curve by plotting the log molecular weights of the protein standards on y-axis against their Rf values on x-axis. Generate the straight-line equation for that line. 4. Next, determine the molecular weights of the first four major protein bands in the rest of the lanes that you measured. From the equation of the standard curve, the molecular weight of an unknown protein can be estimated. Use the equation of the standard curve to calculate the log(MW) of the four bands using in the Rf value for each band as the X value in the equation (you will be solving for Y, the log MW). The inverse log [ =10^(cell#) ]of that number will be the molecular weight of that band. In different samples are four major proteins different? 4 Appendix I: Downloading and Installing Image J. If you are using your own computer or another one on campus rather than one in the lab, you must download and install Image J. This is a simple process that takes only a few moments. Remember that if you are using another computer on campus, you should save your data to your network space before you quit your session or the data will be lost. 1. For Windows: a. Go to rsb.info.nih.gov/ij/download.html b. Under Windows, click on “Download ImageJ 1.39 bundled with Java 1.6.0_05.” c. Select “Save to Disk” and select the desktop as the destination. d. It will take a few moments to download. When it is finished, double click on the .exe file that now appears on your desktop: ij139-jdk15-setup.exe. Follow the instructions for installation. e. The Image J microscope icon should appear on your desktop. Double click it to launch the program. 2. For Macs: a. Go to rsb.info.nih.gov/ij/download.html b. Under Mac OS X, click on “ImageJ 1.39.” c. Select “Save to Disk.” d. An icon labeled ij139.tar.gz with appear on the desktop; double click it, then double click the ImageJ folder that will appear. e. Double click the ImageJ microscope icon to launch the program. 5