Correctly aligning Western blots to 2D gel images
What is alignment?
Alignment takes two images and overlays them in the same coordinate space so that a spot, on the image to be aligned, will be in the same location as the matching spot on the target image.
Understanding when alignment is correctly implemented is easy in SpotMap as there are generally visual clues the software that you can look out for to confidently know that the images are ready for further analysis. However, alignment in SpotMap can be changed and corrected at any point in the analysis. There are three ways check the alignment is correct:
Visually check the direction of the vectors
When you manually or automatically align the images you will see a circle with a line indicting the direction of the alignment, we call these vectors. The direction of the vectors are normally a good indicator that alignment has or hasn’t been done correctly. Vectors in a similar location should all be pointing in the same direction or thereabouts.
Review the alignment if your vectors don’t follow the similar location.
Check the grid for warping
When your images are aligned you can overlay a grid in alignment. If the grid looks unusually warped in some areas you should manually review the vectors in that area.
How can I aid Alignment?
If you can spot any inaccuracies you can aid the alignment with manual vectors this method can be used in addition to automatic alignment or on its own. It ensures that all spots fall within the outlines.