1. 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:
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 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