Procedures

  Antibody validation

All the antibodies for newly identified basement membrane proteins generated in our project were affinity-purified and their specificities were validated using the following criteria:

1. Specifically recognizing their antigenic fragment in ELISA and Western blotting.
2. Specifically recognizing their full-length recombinant protein expressed in mammalian cells in ELISA and Western blotting.
3. Losing their immunohistochemical reactivities upon adsorption with antigenic fragments and with the full-length recombinant proteins.
4. Producing identical staining patterns to those of corresponding antibodies raised against non-overlapping fragments.

All the antibodies used in the database must meet criteria 1, 2 and 3 or 4. Criteria 3 and 4 provide a basis for an evaluation and scoring of antibody validation: antibodies that met criteria 3 but not 4 were given the validation score "Medium" and the antibodies that met criteria 4 were given the score "High". If the antibodies met neither criteria 3 nor 4, they were given the score "Low".

We also produced polyclonal and monoclonal antibodies against known basement membrane proteins. All the polyclonal antibodies were affinity purified. The specificities for the antibodies against known basement membrane proteins were validated based on the similarities of immunohistochemical staining patterns to those published in localization literature(s) (criteria 5) and criteria 4 indicated above. The validation score "Medium" was given to the antibodies which displayed almost similar immunohistochemical staining patterns shown in the published literature(s) in certain tissues. The validation score "High" was given to the antibodies which also met criteria 4. If the staining patterns of the antibodies were not ensured by the published results or criteria 4, the score "Low" was given.

For commercially available antibodies immunohistochemical conditions were determined to obtain reasonable results basically according to the manufacturer's protocols when they have been available. The validation scores were given on the basis of the similarities of the staining patterns to those described in the published localization literatures.

Each validation score is given in the "Protein list" and "Antibody info" pages.