Number Scoring of Phosphospecific AfCS Antibody Database: New Web-based Version Becky Fulin, Lonnie Sorrells, Robert Sinkovits*, Ruth Levitz, and Heping Han The Alliance for Cellular Signaling: The Antibody Laboratory (University of Texas Medical Center at Dallas) and the Bioinformatics and Data Coordination Laboratory (University of California at San Diego)* Abstract The Antibody Laboratory has tested numerous commercially available antibodies for their utility by Western immunoblotting. We have tabulated results in database form and posted the information on the Web in a new interactive version. To date the database includes information on over 300 antibody preparations. Our goal is to provide the signaling community with an immediate resource on our experience with commercial antibodies and to supply updates as we progress. We have posted a Brief Communication to instruct readers on how to navigate the database and to provide information on how we judge the utility of the antibodies that we test. How to Find the Database Go to AfCS website at: www.signaling-gateway.org Click on Links to the database and the brief communication are located on the right hand side of web page. Navigating the Antibody Database The database is organized into three levels: Level 1: Index of Antibody Targets Level 2: List of Antibodies to Chosen Target Level 3: Antibody Tables A separate table is generated for each antibody tested Each row in a table represents results from a separate experiment You may refine your search at any level Level 3: Example of Antibody Tables Red color indicates a Best Score of 1 Each row represents a single experiment Antibody name and Catalog number Measured molecular weight Blue color indicates a Score of 3 Grey color indicates a score of 4 Green color indicates a Score of 2 Expected molecular weight The Search Engine Categories available to refine search: Vendor Score Phosphospecific Cell type Cell Treatment For Example you can search for: All CST phosphospecific antibodies that scored 1 tested with IO4 treated RAW 264.7 cells Level 1: Index of Antibody Targets Scoring Background Evidence of Antibody Phosphosensitivity Number Scoring of Phosphospecific Antibodies We have scored the background in our experiments with none, +, ++, or +++, in order of increasing background that we observed. None + ++ +++ < Many of the antibodies we test are targeted to a specific site of phosphorylation (phosphospecific). We test these antibodies with lysates from cells exposed to an appropriate ligand or phosphatase inhibitor, which is anticipated to increase phosphorylation of the protein. The best antibodies allow us to see a difference in intensity of the correctly sized band when comparing cells in the basal or stimulated states; this is considered evidence of phosphosensitivity. 4 (grey) = Results do meet criteria listed above. LIF treated ISO treated Anti-IgM treated 1 (red) = Unambiguous band(s) of expected molecular weight, very little or no background. Phosphosensitivity is evident. These antibodies are of quality necessary for multiplex Western blotting. Number Score 2 3 1 2 (green) = Band of expected size is observed but it is weak or accompanied by additional bands. Phosphosensitivity is evident. 3 (blue) = Prominent band(s) of expected size(s), but we do not see evidence of phosphosensitivity in the experiment. Example Use of Search Engine Level 2: Antibodies to Chosen Target Antibody names are linked to summary tables of experimental results (level 3). Catalog numbers in colored font are hot-linked to antibody information page (for antibodies from AfCS collaborator, Cell Signaling Technology). Readers should be aware that poor results (those scored 4) do not necessarily mean that the antibody is without utility. It may mean that the conditions, in which the antibody was tested, were not suitable. Alternative conditions such as different cells or cell treatment may give better results. These points are made in our Brief Communication. A Word of Caution