![imagej western blot quantification citation imagej western blot quantification citation](https://i.ytimg.com/vi/9fipZozYuos/maxresdefault.jpg)
Inside each window will be a mountain, unless there was more than one band in the lane, in which case there will be an equivalent number of mountains. ImageJ will open a new window that has windows stacked horizontally, with one window for each of your lanes. After you have dragged the last rectangle over the last lane, indicate that you are finished by selecting: Analyze > Gels > Plot lanes (or command -3).(Note: You don't have to be careful about the height of the boxes, as ImageJ will automatically align the boxes.) Repeat until all the lanes have been highlighted with a rectangle.Indicate this is another lane by selecting: Analyze > Gels > Select next lanes (or command -2) Using the mouse, click inside the rectange and drag (a second box will drag) to the second lane.Indicate that this is the first lane of the gel by selecting: Analyze > Gels > Select First lane (or command - 1) If there are multiple bands in the lane, make sure there is white space between the bands. Include some white space above and below the band (or bands). Use the rectangle tool to create a vertical rectangle in the first lane.You have to do this again, because the powerpoint file saves it as an RGB tiff file. Convert the image to 8-bit (Image > Type > 8-bit).Open the TIFF file you created above in ImageJ.Powerpoint saves a folder with the filename, and then saves each slide as slide1.tiff, etc. Save the file as a TIFF file using Powerpoint save as command.Do not change sizes or adjust the images in anyway.Note: You can also label everything, put in ladder images and other things, as long as you leave white space around the images.Line them up, leaving white space in between each one.Open a Powerpoint file and drag all your image files into powerpoint.Repeat this process using your loading control image file, if it is a different file.Save the file, and repeat with the other file copies, if you have more than one set of bands on the same original image.You should now have a very small file with a rectangle that only includes the bands you want to measure Try to draw the rectangle as small as possible around the horizontal line of bands. Crop the image by highlighting the bands you want to measure using the Rectangle tool.Enlarge the image using the Magnifying Glass tool so you mainly just see the bands you want to measure.If there are more than one set of bands that you want to analyze, make as many copies of the file as there are bands that you want to analyze.Select at least 6 gridlines to help you judge whether or not the bands are horizontal.Select the angle you want (it will rotate clockwise).Rotate the image so the bands are lined up horizontally (Image > Tranform > Rotate).If you have black background and white bands, invert the image (Edit > Invert).Save the files as TIFF file using a different name than the original.Figure 2 shows a stylised western blot of increasing concentrations of protein, and the “signal intensity” as measured by a commonly used software-in this example the last five concentrations gave the same intensity measurement despite representing very different amounts of protein. This represents a general problem of quantifying western blots with simple image analysis software, which may be unable to discriminate between similar-looking bands that have fallen off the end of the linear scale. The chemiluminescent film was saturated, so the higher level of tubulin in the wild type was not reflected when the intensity measurements were taken: actually when the same amounts of sample were loaded, there was no change in expression of Protein X in the two conditions. In fact, the gel for the wild type was accidentally loaded with more of the sample. However, although the two tubulin controls look the same-and give the same intensity measurements using a simple image analysis tool-they do not represent the same underlying expression.