![]() ![]() The calculated densities are scaled relative to the standard sample from lane 1. ![]() Alternatively, you can go to the Table tab and click on the Launch Spreadsheet button, which will create and open a new Excel file with your data in it (Figure 11).įigure 13. To begin with, you can simply highlight all of the data rows in the Shapes tab at the bottom of the window, copy them, and paste them into Excel. With the raw signal values, I can now calculate the relative density of all of the sample bands relative to my Human Hsp70 sample standard. See this explanation of why this should concern you and what to do about overexposure. Incidentally, if your bright bands return with a value of Infinity, this is an indication that your blot image was overexposed. Either way, the important point is that the higher density bands with more protein in them have larger absolute values (very negative in my case). If you’re dealing with a gel image where the bands are lighter than the background, your values should come out positive. This is just a function of the way the program deals with the grey values stored in my tiff. You’ll note that my values are all negative. All of the bands have been selected now.įor this basic analysis, the quantity I’m concerned with is the Signal column. I draw the first rectangle large enough to encompass the band in the first lane, but also making sure that it will be large enough to also cover the same band in any of the remaining lanes (Figure 7).įigure 10. There are additional options like the auto Add Rectangle, but I keep finding that I have to manually adjust the auto-rectangles, so it’s easier to just start with a custom rectangle. This tab provides the tools to select the bands you want to analyze. The next step is to switch to the Shapes tab near the top of the main Image Studio window. The remaining lanes are samples of Hsp70 from rocky intertidal limpets that were exposed to various levels of heat stress. My example blot ( here’s a copy of the original file) includes a sample of Human Hsp70 protein purchased from a commercial provider, which I was using as my sample standard in the far left lane on this project. You might be working with a fancy laser scanner image, or a digital image from a gel camera, but the concepts are similar. In this case, I’m working with a tiff image from a western blot that was digitally scanned off of a piece of x-ray film, because I’m old school, or just old. Returning back to the main Image Studio window, the western blot image is now displayed properly. You can adjust the image display further in the next window. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |