In my last post, I shared how Progenesis SameSpots can compare proteins detected on Coomassie stained 2D gels and Western Blots. But comparing secondary stained images with 2D gels has applications beyond characterising proteins of interest in differential expression analysis.
For example, 2D gel electrophoresis can be used at several stages of recombinant protein production:
- Characterise isoforms of recombinant proteins
- Confirm purity and identity of contaminants
- Characterise antibodies as suitable candidates for ELISA assays
The final application has been demonstrated by Kendrick Labs, who characterise anti-HCP antibodies for reliable quantitative ELISA assays. These are then used to measure HCP contamination in biologic drug production. As I discovered, the tag feature used alongside the 2D montage could be used to achieve this simply in the current version of Progenesis SameSpots.
After aligning a 2D gel and Western Blot and analysing both images together, you end up with the same population of spots across both images.
In the View Results section of the workflow, as you work down the list of detected spots, each one is displayed as it appears on both the 2D gel image and the Western Blot. Using the Progenesis SameSpots approach means you are directly comparing the same coordinate space on both images. This overcomes the challenge of comparing signals from two different images with very different sensitivities and protein specificity. At the same time you can also split or merge spots and be sure that the same edit is applied consistently to both images to maintain a common spot map.
In the example, above I tagged a spot detected by antibody signal on the Western Blot, which appears undetected by Coomassie Blue staining on a 2D gel. I could also have tagged spots present on the 2D gel that were not targeted by the antibody.
At the end of this process, simply apply tag filters to show all spots that are tagged as unique to the Western Blot. The number of tagged proteins is displayed so you can express this as a % of the total spot population created from both images as part of the SameSpots analysis process.
This can provide an index of how good an antibody is for detecting host-cell proteins in a purified recombinant protein sample.
How does Progenesis SameSpots achieve this?
Highly accurate pixel-level alignment means you can directly compare differentially stained images. The western blot image (green) has been aligned to the 2D gel image (magenta).
|Comparing two images that are very different, not only in terms of spot patterns but also intensity, is a big challenge.
Progenesis SameSpots applies highly accurate pixel-level alignment so you can directly compare 2D gels with secondary stained images. It’s an integral part of the analysis process, so you can easily try it on your own images.