How to Align Western blots to 2D Gel Images for HCP antibody analysis

What is bit depth?

Bit depth is the number of bits used to represent the grayscale (intensity level) of each pixel in an image. Greater bit depth allows a greater range of shades of grey to be represented by a pixel. For example, an 8-bit grayscale image file stores 256 shades of grey for each pixel, while a 16-bit image file has 65536 possible grayscale values for each pixel. The following table indicates the possible grayscale levels available for the types of images commonly used for gel image analysis.

Bit Depth Intensity Levels
8 256
10 1024
12 4096
16 65536

In reality, the images displayed on the computer screen will only be represented in 256 shades of grey, and so an 8-bit image will look identical to a 16-bit image by eye. However, image analysis software can distinguish between the different levels of grey.

How can I capture my images?

As a rule, the more levels of grey represented in an image, the better the ability to differentiate low abundance spots from the background, and the greater the quantitative accuracy therefore we recommend capturing as 16 bit images. This is further illustrated in the video, comparing spot detection in an identical area on the same 2D gel, captured at 8-bit and 16-bit.

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