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It’s like, how much more black could this be? And the answer is none. None more black.

– Nigel Tufnel, lead guitarist of Spinal Tap.

None more black (or none more white) is a real concern when you’re talking about imaging western blots. If you only want to verify the presence of a protein, you can stop reading here, because even an overexposed image will tell you that there is some protein present in your sample. But an overexposed image of a western blot is essentially useless when you’re … Continue Reading

Before I lose this file again: StartupMacros.txt This is a text file, based on the original StartupMacros.txt file that came with ImageJ, that can be dropped in the ImageJ/macros folder that is created when you install ImageJ. Start up ImageJ, and if this macro doesn’t automatically run, go to Plugins>Macros>Startup Macros to load it. It installs a few more drawing tools on the ImageJ toolbar and creates some keyboard shortcuts for the following tools:

• Line tool – press ‘l’
• Brush tool – press ‘b’
• Eraser tool – press ‘e’
• Wand tool – press ‘w’

I either found or created some code … Continue Reading

Following on this earlier post concerning the retrieval of data from figures in published papers with R, here is a method for doing the same with ImageJ. This is a freely available program that should run on Windows/Mac/Linux, just like R. To extract data points from a figure, you’ll need the Figure_Calibration plugin developed by Frederic V. Hessman, available here. Download the Figure_Calibration.class file from that page and save it to your ImageJ/plugins/ directory. Then load up ImageJ.

[caption id=”attachment_1051″ align=”aligncenter” width=”400″ caption=”The example figure that we wish to retrieve data from. We can use … Continue Reading

ImageJ is easy to modify if you know a bit of Java. I don’t know any Java. But here’s what I did to add keyboard shortcuts for the paint brush tool and the line selection tool. The code is based off this help file from the ImageJ website: http://rsbweb.nih.gov/ij/docs/guide/userguide-32.html

The first step was to figure out to refer to the line tool and paint brush tools. I did this by going to Plugins>Macros>Record and clicking on the line tool and the paint brush tool in succession. The numeric codes for those tools were revealed in the macro recording window … Continue Reading

The following is a tutorial on using the freely available ImageJ program from the National Institutes of Health for estimating the area of a leaf or algae blade (or anything else). The process is straightforward once you understand the various steps.

You must begin with a photograph of your object, flattened and dried off as much as practical. Include a clearly visible ruler or other measuring device in the picture. Ideally you will photograph your dark leaf or blade on a light background to maximize the contrast between the object and the background.

[caption id=”attachment_1009″ align=”aligncenter” width=”300″ caption=”The original picture, … Continue Reading

The following information is an updated version of a method for using ImageJ to analyze western blots from a now-deprecated older page. If you need a peer-reviewed citation for the methods outlined below, you may cite  Stael, S., L.P. Miller, Á.D. Fernández-Fernández and F. Van Breusegem (2022). Detection of Damage-Activated Metacaspase Activity by Western Blot in Plants. Plant Proteases and Plant Cell Death: Methods and Protocols. M. Klemencic, S. Stael and P.F. Huesgen. New York, NY, Springer US: 127-137 https://doi.org/10.1007/978-1-0716-2079-3_11

If you’re looking for a more comprehensive workflow option for your western blot … Continue Reading