Western Blotting

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Wed May 5 14:29:37 EST 2010


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



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