MS-Powerpoint-Word-Count-Frequency.png' alt='Keil C Programming Tutorial Pdf' title='Keil C Programming Tutorial Pdf' />Blog Archive Analyzing gels and western blots with Image. JThe following information is an updated version of a method for using Image. J to analyze western blots from a now deprecated older page. If youre looking for a more comprehensive workflow option for your western blot analyses, please visit my tutorial on using Image Studio Lite, a free software package from LI COR Biosciences. The Image Studio Lite software can be downloaded for free from www. In addition to the functionality described below for Image. J, Image Studio Lite provides additional data management features along with annotation tools for producing publication ready images of your westerns. A pdf copy of this page is available. Image. J http rsb. This tutorial assumes that you have carried your gel or blot through the visualization step, so that you have a digital image of your gel in. If you are scanning x ray film on a flatbed scanner, make sure you use a scanner with the ability to scan transparencies i. See the references at the end of this tutorial for a discussion of the various ways that you can screw this step up. This also assumes that you didnt overexposed your blot image when you produced it. For an explanation of why overexposure will ruin your results, see this page. The method outlined here uses the Gel Analysis method outlined in the Image. J documentation Gel Analysis. You may prefer to use it instead of the methods I outline below. There should be very little difference between the results obtained from the various methods. This version of the tutorial was created using Image. J 1. 4. 2q on a Windows 7 6. Open the image file using File Open in Image. J. 2. The gel analysis routine requires the image to be a gray scale image. The simplest method to convert to grayscale is to go to Image Type 8 bit. Your image should look like Figure 1. Figure 1. A fabricated western blot image opened in Image. J. The information along the top of the image indicates that the image is currently in 8 bit mode, using an inverting LUT look up table. The inverting LUT ensures that dark bands will be recorded as higher density values. Choose the Rectangular Selections tool from the Image. J toolbar. Draw a rectangle around the first lane. Image. J assumes that your lanes run vertically so individual bands are horizontal, so your rectangle should be tall and narrow to enclose a single lane. If you draw a rectangle that is short and wide, Image. J will switch to assuming the lanes run horizontally individual bands are vertical, leading to much confusion. UPyqx_UDts/0.jpg' alt='Keil C Programming Tutorial Pdf' title='Keil C Programming Tutorial Pdf' />Figure 2. The first lane has been selected with the Rectangular Selections tool. After drawing the rectangle over your first lane, press the 1 Command 1 on Mac key Command 1 on Mac or go to Analyze Gels Select First Lane to set the rectangle in place. The 1st lane will now be highlighted and have a 1 in the middle of it. Use your mouse to click and hold in the middle of the rectangle on the 1st lane and drag it over to the next lane. You can also use the arrow keys to move the rectangle, though this is slower. XXo_4m46qY/0.jpg' alt='Keil C Programming Tutorial Pdf' title='Keil C Programming Tutorial Pdf' />Kilauea Mount Etna Mount Yasur Mount Nyiragongo and Nyamuragira Piton de la Fournaise Erta Ale. Copyright 2014 ARM Limited or its affiliates. All rights reserved STMicroelectronics Discovery STM32F407 Lab with ARM Keil MDK toolkit www. Center the rectangle over the lane left to right, but dont worry about lining it up perfectly on the same vertical axis. Image J will automatically align the rectangle on the same vertical axis as the 1st rectangle in the next step. Press 2 Command 2 on Mac or go to Analyze Gels Select Next Lane to set the rectangle in place over the 2nd lane. A 2 will appear in the lane when the rectangle is placed. Repeat Steps 5 6 for each subsequent lane on the gel, pressing 2 Command 2 on Mac each time to set the rectangle in place Figure 3. Figure 3. Place the rectangle over each lane, pressing 2 each time to set the rectangle in place. After you have set the rectangle in place on the last lane by pressing 2, press 3 Command 3 on Mac, or go to Analyze Gels Plot Lanes to draw a profile plot of each lane. Figure 4. The profile plot from the example western blot. The profile plot represents the relative density of the contents of the rectangle over each lane. Mi Familia Rapidshare more. The rectangles are arranged top to bottom on the profile plot. In the example western blot image, the peaks in the profile plot Figure 4 correspond to the dark bands in the original image Figure 3. Because there were four lanes selected, there are four sections in the profile plot. Higher peaks represent darker bands. Johnny Cash Discography Rapidshare. Wider peaks represent bands that cover a wider size range on the original gel. Images of real gels or western blots will always have some background signal, so the peaks dont reach down to the baseline of the profile plot. Figure 5 shows a peak from a real blot where there was some background noise, so the peak appears to float above the baseline of the profile plot. It will be necessary to close off the peak so that we can measure its size. Figure 5. Real western blots often have some background noise, so the peak doesnt reach down to the baseline perfect white. Choose the Straight Line selection tool from the Image. J toolbar Figure 6. For each peak you want to analyze in the profile plot, draw a line across the base of the peak to enclose the peak Figure 5. This step requires some subjective judgment on your part to decide where the peak ends and the background noise begins. Figure 6. Use the straight line tool to close off the base of each peak of interest. Figure 7. The peak from Figure 5 is shown with a line drawn across the base of the peak to enclose the area of the peak. Note that if you have many lanes highlighted, the later lanes will be hidden at the bottom of the profile plot window. To see these lanes, press and hold the space bar, and use the mouse to click and drag the profile plot upwards. When each peak has been closed off at the base with the Straight Line selection tool, select the Wand tool from the Image. J toolbar Figure 8. Figure 8. Choose the Wand tool to highlight each peak of interest on the profile plot. Using the spacebar and mouse, drag the profile plot back down until you are back at the first lane. With the Wand tool, click inside the peak Figure 9. Repeat this for each peak as you go down the profile plot. For each peak that you highlight, measurements should pop up in the Results window that appears. Figure 9. Click inside the 1st peak of interest with the Wand tool. The peak should be highlighted and a measurement should pop up in the Results window. When all of the peaks have been highlighted, go to Analyze Gels Label Peaks. This labels each peak with its size, expressed as a percentage of the total size of all of the highlighted peaks. The values from the Results window Figure 1. Edit Copy All in the Results window. Paste the values into a spreadsheet. Figure 1. 0. The output from selecting peaks in the profile plot and labeling the peaks. In this case, the results are from selecting the upper band in each of the 4 lanes in the example western blot from Figure 1. Note If you accidentally click in the wrong place with the Wand, the program still records that clicked area as a peak, and it will factor into the total area used to calculate the percentage values. Obviously this will skew your results if you click in areas that arent peaks. If you do happen to click in the wrong place, simple go to Analyze Gel Label Peaks to plot the current results, which displays the incorrect values, but more importantly resets the counter for the Results window. Elektor March April 2. Edition by Elektor. Elektor March April 2. Edition   Published on Feb 2. Look inside Elektors March April 2. Edition. In this edition 6 Labs Projects, 1 Programming Course, 6 New Modules and PCBs, 1. Readers. See More.