Dear Yvonne, basically, you need to run standards in different amounts (like serial dilutions) and build a calibration curve. This needs to be done for every protein of your interest. Relative calibration, i.e. dilutions of a reference sample, eg a cell lysate, can do the job.
ImageJ has calibration functions (thank's to Wayne Rasband's generous support). If you work with X-Ray films, the calibration will become very tedious, because the dynamic range is less than 2 decades and it's hard to expose the film in a way that all bands of interest are in the slope-range of the standard(s). Thus, I hope you have access to a more sophisticated instrument like an infrared fluorescence scanner with a higher dynamic range. Still a lot of manual work with ImageJ, but you will obtain data you may do statistics with. This topic also appears in the ImageJ mailing list from time to time, so you might find some more information there at https://list.nih.gov/cgi-bin/wa.exe?A0=IMAGEJ when you search for "western". Reagrds, Wo _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
