Will this antibody cross-react?
When selecting an antibody, it's important to ensure it is specific to the target and does not cross react. Find out what you need to know to minimize cross-reactivity.
Avoiding cross-reactivity with pre-adsorption or antibody fragments
Checking potential cross-reactivity for similar proteins
Principles
When selecting an antibody:
- Make sure it's validated to bind to the target. Check the 'overview' and 'target' sections of the datasheet.
- If chosing a secondary antibody, make sure it's raised against the primary antibody in a different species, e.g. a mouse primary might require a donkey anti-mouse secondary.
Avoiding cross-reactivity with pre-adsorption and fragments
- If a secondary antibody has been pre-adsorbed against potentially cross-reactivie proteins, it will not react with those proteins. This increases specificity and minimizes cross-reactivity.
- Using F(ab) and F(ab')2 antibody fragments rather than full immunoglobulins can further minimize potential cross-reactivity between antibodies and cell-surface receptors.
Checking potential cross-reactivity for similar proteins
If you have the immunogen sequence of your antibody, you can check the sequence alignment of the immunogen with other proteins the antibody could react with. There are several websites that provide tools for calculating the percentage alignment, which are accessible from the EMBL-EBI website.
When comparing the immunogen sequence and another protein sequence, an aligment score of 85 % or higher indicates the antibody may cross react; the percentage alignment should ideally be much lower than this.