What are your results? How it works and does it change the standard wb protocol?
Thanks!
Hi Maria,
please find a good tutorial here (http://imagejdocu.tudor.lu/doku.php?id=video:analysis:gel_quantification_analysis ) but do NOT use the background substraction - that will change inividual pixel values in conjunction of the adjacent pixel information.
Best Soenke
Thank you for the fast reply! Do you need a special detection system for this purpose?
Hi Maria,
I would recoment a normal chemilumineszenz box for Westernblot (try google image search) - they are usally equipped with a 12, 14, or 16 bit black and white camera. And you will optain raw 16bit black and white (tiff) images from the camera.
If you only have a dark room and X-Ray film try to scan them with a transmittedlight scanner and to export the image a a 16bit black and white tiff, but you will lose a the higher dynamic range that the camera is able to provide. I heard that film is only providing up to 100 gray scales where a 16bit cmaera image can give you up to 65T important is not to oversaturate the film as well as while scanning the film (to total black).
take also a look at:
http://www.piercenet.com/method/overview-western-blotting#detectionmethods
http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf
http://www.pottslab.org/protocols/Protein%20Biochemistry/protein_transfer_guide.pdf
There are also the pros and cons discussed for different detection methods.
Best Soenke
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