![]() How to avoid cross-reactivity during immunohistochemistry/immunofluorescence experimentsĬross-reactivty might occur in immunostaining assays in tissue samples, for example while working is mouse tissues with mouse-derived antibodies. 10442-1-AP has a 71% homology with Sheep, and therefore a strong likelihood of reactivity. Step 3: Search for the organism (use the species' Latin name) you want to check homology with (under "Standard"). Step 2: Paste sequence into the NCBI-BLAST "query sequence" box. Step 1: Locate the immunogen sequence for the antibody (link to immunogen sequence can be found under "Product information"). Working example: What is the likelihood of Proteintech's P53 polyclonal antibody ( 10442-1-AP) cross-reacting in sheep samples (Ovis aries)? As mentioned above, you can use NCBI-BLAST and specifically select for your species of choice. It depends on the extent of protein sequence similarity between the immunogen and the potential cross-reactive protein sequence. Anything over 60% has a strong likelihood of cross-reacting, but will need to be verified in an assay and sample specific manner.ĭoes my antibody cross-react with other species? What is the “right number” for homology to predict cross-reactivity?īased on our extensive experience in antibody development and working with thousands of customers, the magic number for almost guaranteed cross-reactivity is 75% homology with the immunogen sequence. 95% of Proteintech's antibodies are raised against recombinant, long-length human proteins, meaning that they have an increased chance of cross-reactivity in non-human models such as mouse, rat, drosophila and zebrafish. Conversely, if you are looking for an antibody to work in other species, use a polyclonal antibody against a recombinant, long-length protein. Therefore if you suspect your antibody of cross-reacting negatively, consider switching to a monoclonal antibody (a homologous IgG antibody population only recognizing one epitope). Remember that polyclonal antibodies have a higher chance or cross-reactivity due to the fact that they recognise multiple epitopes along the immunogen sequence. Cross-reactivity is not always a negative quality, for example cross-reactivity of an antibody for a target across species allows the same antibody to be used in multiple model organisms. Here, you can check for an antibody's potential cross reactivity against that of similar proteins or in other species. This can be achieved by pair-wise sequence alignment using NCBI-BLAST. Thus, testing an antibody for cross-reactivity with closely related proteins is a to ensure accurate results.Ī quick and easy check for antibody cross reactivity is to assess the percentage homology of the antibody immunogen to that of other similar proteins. This is often the case when two antigens have similar structural regions that the antibody recognizes.Ĭross-reactivity can invalidate the results of an experiment and has negative impacts on scientific reproducibility. Cross-reactivity between antigens occurs when an antibody raised against one specific antigen has a competing high affinity toward a different antigen, therefore the antibody is able to recognize a protein which is different to the one it was raised against. An antibody has a specific amino acid sequence (the Fab region) that dictates its affinity for a specific antigen.
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