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

A 2D gel image is compared to the Western blot and a coverage calculation is determined (total protein spots detected by the antibody). Due to the nature of 2D electrophoresis some variation will be introduced during the gel running, blotting or imaging process. The spot locations on the images will have to be matched, in SpotMap we call this Alignment.

Imaging Techniques

Warping

What is warping?

Warping techniques aim to remove the positional variation but are only accurate to the spot level.

How is alignment different to warping?

Warping and alignment both aim to remove the positional variation introduced during the gel running, blotting and imaging process. However alignment is accurate to the pixel level.

Spot Matching

What is spot matching?

Spot matching techniques detect spots on each of the images separately then spots are matched between them.

Why is alignment better than spot matching techniques?

Using separate spot detection on each image and then matching each spot between the images is very inefficient and inaccurate. Alignment makes it possible to overlay spots from one image to another, placing them in the correct location.

Alignment

What is alignment?

Alignment is a key feature within SpotMap image analysis software which allows removal of this positional variation, providing easy comparison of spot patterns between 2D gel and Western blot images.

Using DIGE technology is way to forgo the alignment/spot matching process altogether. However, for low MW proteins there may be a shift due to the different dyes. In this case Alignment can be used.

Alignment in SpotMap

The following video on the right visualizes how you can align your images in SpotMap in 5 simple steps. It is designed for quick and easy comparison even on substantially different images.

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We’ve created some more useful documents discussing alignment

How do I check my Alignment is correct?

How do I know that I have completed Alignment?

The image capture effect on your image analysis results