Hello everyone,
I have TIFF images of western blots that were acquired using Syngene chemidoc machine. I have converted TIFF files into 8-bit images using ImageJ (as there was a error opening the files in Imagelab). Then I opened the files in Imagelab and started to analyze. The peaks of the band, in the lane profile are inverted. I tried to save the 8-bit file as .scn file and then open it, but still the peaks look inverted.(pls see the attached file)
Did anyone else face this issue?
Please suggest a solution.
Hi Razia,
There is only the peak image, and not the raw image of your gel.
Nonetheless, I think you see negative peaks because Western Blot Gel Images are usually dark bands on a light background.
8-bit images are coded from 0 (black) to 255 (white), then bands have lower values than background, resulting in this "inverted" peak.
If you want to have upright peaks, you can invert the image under ImageJ (Edit>Invert or Edit>LUT>Invert LUT depending on the version you have).
Cheers
Oliv
Hii Oliv,
Thank you so much for your suggestion. It worked!.
Attached is the western blot image.
Best Regards,
Razia
I have one more question Olivier Catrice . The analysis done with inverted peaks is meaningless, am I right?
Hi there,
What Olivier Catrice said is perfectly correct; and no, the results aren't meaningless. They're just inverted.
Assuming you are working with 8-bits, the value of each pixel for "positive peaks" is something like:
Value "positive peak" = 255-Value "negative peak"
So a value of 0 (Darkest possible pixel in images like the one attached) will yield a value of 255.
Following this reasoning, a value of 255 (Brightest possible value in images like the one attached) will yield a value of 0.
So, in the first image attached (the "inverted peak") your background levels are around 55 (255-200) and your peak levels would be something around 100 (255-150),
Cheers,
J
Hi Razia,
J is right, results obtained with inverted peaks have the same significance than upright peaks.
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