Antibodies are validated using context dependent models and cell lines. This way, the type of tissue CST tests the antibodies on, will likely be the same as the ones you are using for IHC. The types of validations required differs depending on the antibody’s application. See what CST scientists have to do for each clone, to meet the standards for an antibody to be sold:
Western Blotting Validations:
siRNA knock down, Phosphatase treatment, multiple cell lines, find out the
in-depth validations for WB antibodies.
TMAs, blocking peptide, xenograft models, paraffin and fresh frozen, find out the
in-depth validations for IHC antibodies.
Verification of subcellular localization, activator/inhibitor treatment, threshold signal-to-noise ratio, find out the
in-depth validations for IF antibodies.
Pdx1 Antibody #2437: Confocal immunofluorescent analysis of normal rat pancreas using Pdx1 Antibody #2437 (green, upper) or Insulin (C27C9) Rabbit mAb #3014 (green, lower). Keratin filaments were labeled with Pan-Keratin (C11) Mouse mAb (Alexa Fluor® 647 Conjugate) #4528 (blue). Red = Propidium Iodide/RNase #4087 (fluorescent DNA dye).
Flow Cytometry Validations
Isotype controls, Titrations, localization of antibody for intracellular stains , find out the
in-depth validations for Flow antibodies.
A comparative analysis of the performance of a phospho-Stat5 antibody from CST (#4322) with a phospho-Stat5 antibody from an alternative vendor. When looking at a cell line positive for phospho- Stat5, both antibodies enable detection. While the non-CST Ab clone gives rise to a brighter signal by flow cytometry (A) (bar graph on the left), which is dampened by imatinib treatment, it exhibits incorrect sub-cellular localization when examined by IF (B). This is particularly evident when cells are treated with EGF to enhance the strength of the signal. This suggests that only the CST antibody clone for phospho-Stat5 detects its target specifically, correctly localizing to the nuclear compartment of the cell.