the western blots were visualized using Amersham Imager 600 gel documentation system (GE Healthcare Bio Sciences AB), four different images were taken consecutively and automatically
question 1:
In image 1, how come there is a white region inside the protein blots? Is that overexposure?
question 2:
Comparingimage 1 and 2, how come blots in image 2 are so much stronger than image 1, and there are no white region in the blots of image 2?
It appears to me that image 1 was a very short exposure and image 2 was a much longer exposure.
The short exposure emphasizes the slight variation in intensity across the bands, with the white regions representing areas of lower intensity. I would consider image 1 to be underexposed.
The much longer exposure of image 2 may have saturated many of the pixels. The software should be able to show you whether any pixels are saturated. Once pixels saturate, they can't get any darker. This allows the areas of lower intensity to "catch up" to the areas of higher intensity. This image may therefore be overexposed.
A good practice for quantitation is to take images at each of several times as the exposure accumulates and to use the darkest image at which none of the pixels is saturated. This gives the maximum dynamic range of intensities.
Hi, Suxiang Chen.
"Question 1:
In image 1, how come there is a white region inside the protein blots? Is that overexposure?"
These white bands are probably caused by bubbles. Of course, if the amount of sample loaded is too large (overexposure), the bands will also be reversed.
Please contact me again.
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