CST Antibody Validation Principles
Our scientists are at the bench daily to produce and validate our antibodies, so they have hands-on experience and knowledge of each antibody’s performance.
Specificity + Sensitivity + Reproducibility = CST Validation
Recent reports suggest there are an increasing number of high-impact research studies that cannot be replicated by other scientists. These reports have raised concerns about incorrectly or insufficiently characterized antibodies. At CST, our scientists follow a stringent testing process that involves a combination of assays to provide you with a highly specific and thoroughly validated antibody.
Verifying Specificity, Sensitivity, and Reproducibility
- Analysis of a large panel of cell lines with known target expression levels
- Treatment of cells with appropriate kinase-specific activators and/or inhibitors
- Phosphatase treatment
- Correct subcellular localization or treatment-induced translocation
- Comparison of results with antibody and isotype control to ensure acceptable signal-to-background ratio
- Target-specific signal verified in transfected cells, knockout cells, or siRNA-treated cells
- Blocking with antigen peptide to confirm elimination of specific signal
- Side-by-side comparison of a new lot with previous lots to ensure lot-to-lot consistency
Identifying Optimal Conditions
- Optimal dilutions and buffers predetermined
- Positive and negative control cell extracts specified
- Detailed protocols already optimized
Side-by-side comparison of lots to ensures lot-to-lot consistency.
Western blot analysis of HeLa cells, untreated or treated with IFN-α, comparing lots 1, 2, 3 and 8 of Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb #9145. Note: Signal remains consistent from lot to lot. (Recommended dilution for western blot was changed to 1:2000 with release of lot 3.)
- Lot 1: Jan, 2006
- Lot 2: Jun, 2007
- Lot 3: Oct, 2008
- Lot 8: Apr, 2009
Antibody performance is assessed in relevant mouse models of cancer.
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060: IHC analysis of paraffin-embedded WT (left) and PTEN (-/-) (right) mouse prostate using #4060. Tissue courtesy of Dr. David Guertin, The Whitehead Institute for Biomedical Research, Cambridge, MA.