Using ImageJ to quantify blots - Diamantina Institute - University of Queensland Skip to menu Skip to content Skip to footer UQ Home Contacts Study Maps News Events Library Give now my.UQ The University of Queensland Diamantina Institute Diamantina Institute Site search Search Site search Search Menu Home About News Our people History Our supporters The Translational Research Institute Events Research Research themes Research centres Research by disease Facilities and support services Innovation and commercial development Study Undergraduate Honours Higher Degree by Research Scholarships Visiting Research Students Why should I do my research project at UQDI? Laboratory to patient Clinical trials Clinical research facilities Community and alumni SPARQ-ed Cell and Molecular Biology Experiences Junior 2-day Research Immersions Research Immersion Programs Donate now Contact Using ImageJ to quantify blots Home Community and alumni SPARQ-ed The results of Western Blots can be assessed visually by making comparisons between bands in different lanes. However on occasion, these differences may be subtle and so a more quantitative method should be used. ImageJ is a Java-based image analysis package widely used by scientists in quantitating visual results such as bands on gels or photomicrographs. It is available free for download from the National Institutes for Health (NIH) in the US. This Appendix will take you through the basics of how to use ImageJ in image analysis. Selecting the area to be analysed Open ImageJ from the Desktop or Program Menu. From the File menu, open the image file for the PCNA blot you obtained from the Chemidoc system. Select a rectangular area around the first band using the “Rectangular Select” tool. The area selected should be about twice as tall as it is wide and should be as wide as the widest band on the blot. Press “CTRL” “1” to mark this as the first band. The selection box should be yellow with a blue box with the number “1” inside, indicating that it is the first band selected. Move the mouse cursor so that it sits over one of the box border and changes into an arrow. Drag the box so that it sits over the next band and press “CTRL” “2” to select the next band. Repeat this step for each of the bands, pressing “CTRL” “2” for each subsequent band. With each selection, a small blue box should appear in each of the selection boxes indicating the number of the band (ie. 1-7 in our example). Once all of the bands have been selected, press “CTRL” “3”. This brings up another image which shows histograms indicating the intensity of each of the bands (the larger the histogram, the brighter the band). There should be a histogram for each of your bands – you may need to select the “drag” button and drag the image down to see all of them. Starting with the histogram for the first band, select the “draw line” button and draw a line across the top of the histogram from where it first begins to drop steeply until where it levels out again. Select the “magic wand” button and click anywhere inside the histogram. The selected area should be outlined in yellow and a new window named “Results” should appear which indicates the intensity of the band as a numerical value – the brighter the band, the higher the number. Repeat this process for each of the histograms. You should end up with a numerical value for each of the histograms representing the bands. Once complete, select “File” in the Results window and save the values as a spreadsheet. These values can then be copied into the “Blots Quant” spreadsheet for analysis and comparison. You will need to do this analysis for any other blots you have images for. SPARQ-ed Cell and Molecular Biology Experiences Junior 2-day Research Immersions Research Immersion Programs © The University of Queensland Enquiries: +61 7 3365 1111 | Contact directory ABN: 63 942 912 684 | CRICOS Provider No: 00025B Emergency Phone: 3365 3333 Privacy & Terms of use | Feedback | Updated: 27 Apr 2017 Login
