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UniProt.org: A Free Tool for Comparing Protein Homology

There are some questions that recurrently come to our technical service department. One of them is if our antibodies recognize horse, sheep, pig, monkey, dog, cat, guinea pig, Zebrafish, Drosophila, C. elegans, and more. (While they are not ordered by frequency of request, I’ve at least tried to organize them by decreasing size). Other related questions include for example how similar is a certain protein between two species and how conserved is this protein.
So, the answer to the main question is that we do not perform cross-reactivity tests in-house for our antibodies. So, is there any way to have an idea about possible cross-reaction? What we do is to check for protein homology using online tools that are available to everyone. Our main site of research is the Protein Knowledge Database (www.uniprot.org).

This is a great tool to assess protein homology and you can watch a full tutorial in their site:

https://www.uniprot.org/demos/diabetes

The tutorial is comprehensive, and will show you how to retrieve extra information, such as 3D structure studies of the protein. Here, we can make a short summary, and show you how to use the site to answer the main question:what are the odds our reagent will recognize your target cell?
Go To https://www.uniprot.org/
In conclusion, the higher the homology, the higher the chance an antibody shows cross-reactivity. This is subjective, but for two proteins with 95% homology or more, there is a good chance that the antibody will recognize them both.

So what factors may affect the capacity of an antibody to cross-react?
  • Is the antibody is polyclonal or monoclonal?
    Polyclonals have a higher chance of react because they recognize multiple epitopes on an antigen.
  • Is the epitope highly conserved?
    If the immunogen for the antibody was the full length protein and it has a dominant epitope conserved across species, there is also a good probability that the antibody will cross-react, even if it is a monoclonal and the total homology of the proteins is not very high.
But in the end, all these factors are theoretical, and cross-reactivity has to be experimentally assessed.
Finally, you can check the following pages to see if your antibody has been tested on non-human primates: You can also check our cross-reactivity page as a guideline:

https://www.biolegend.com/cross_reactivity
If you are looking for more specialized reading, here are a couple of links you can check out:

https://www.ncbi.nlm.nih.gov/books/NBK2396/
https://www.bergleslab.com/pdf/Holmseth_et_al_2006.pdf

Have a good day, and if you know of any other tools to evaluate protein homology, let us know by contacting us at: mtam@biolegend.com
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