Antibodies are generally raised against either proteins (partial or full length), or a short peptide sequence, specific to their target protein. However, how do you know whether they bind to the target against which they were raised?
Antibody validation is key!
Below are the most basic validation steps that should have been carried out for your antibody:
Peptide raised antibodies - Immunogen validation
Blast the peptide sequence against the UniProt database to see whether it has identity to its stated target only.
Any proteins where the identity between peptide sequence and protein sequence for the immunogen is 85% or greater could be detected by that antibody.
Unfortunately, the exact peptide sequence is not always available, but the amino acid range surrounding the peptide sequence should be. Blasting the region is also helpful.
All Biorbyt's immunogens are Blasted back to the UniProt database to make sure they detect only the protein(s) they are designed against.
Application validation of antibodies
If an antibody is said to work for Western Blot, or IHC-P, it should have been tested in that application.
Raw validation images should be available for the species application stated.
It is important to see negative as well as positive results.
Additional validations by Biorbyt
Biorbyt ensure each of its antibodies are tested in Western Blot and IHC paraffin embedded sections in Mouse, Rat and if possible Human tissue sections (although this is sometimes difficult depending on the tissue required for the target).
Our Western blot images are shown in their raw form, so you can see species and tissues where the antibody did not detect the target, as well as those where it did.
Remember: validation is key! Follow the steps above to find out whether your antibody will bind to the target against which they are raised.
Check out the following Biorbyt protocols: