Antibody Validation
What does Antibody Validation Mean at Cell Signaling Technology®?
Scientists at Cell Signaling Technology® (CST) follow a stringent Validation Protocol, using a combination of several approaches and applications, to provide you with the highest quality and most thoroughly tested antibodies. This ensures credible and reproducible results with the least expenditure of your costly time, samples, and reagents.
Cell Signaling Technology® Antibody Validation
Testing In A Number Of Applications allows you to choose the antibody that works best in your intended application.
- W
- Western blot
- IP
- Immunoprecipitation
- IHC
- Immunohistochemistry
- IF
- Immunofluorescence-based assays
- F
- Flow cytometry
- ChIP
- Chromatin Immunoprecipitation
Verifying Specificity And Reproducibility ensures that the antibody performs consistently in all applications specified.
- Analysis of a large panel of cell lines with known target expression levels confirms target specificity
- Treatment of cells with appropriate kinase-specific activators and/or inhibitors verifies specificity
- Phosphatase treatment to verifies phospho-specificity
- Correct subcellular localization or treatment-induced translocation verifies target specificity
- Comparison of antibody to isotype control ensures acceptable signal-to-noise ratio
- Target-specific signal verified in transfected cells, knockout cells, or siRNA-treated cells
- Blocking with antigen peptide confirms elimination of specific signal
- Side-by-side comparison of a new lot with previous lots ensures lot-to-lot consistency
Identifying Optimal Conditions saves your precious time, samples and reagents.
- Optimal dilutions and buffers predetermined
- Positive and negative control cell extracts specified
- Detailed protocols already optimized
Lot-to-Lot Testing
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
Isotype Control
Comparison of signal to isotype control is used to estimate the nonspecific binding of primary antibodies.
Rabbit (DA1E) mAb IgG XP® Isotype Control #3900: Flow cytometric analysis of SH-SY5Y cells using CREB (D76D11) Rabbit mAb Antibody #4820 (blue) compared to concentration matched #3900 (red).
Mouse Models
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.
