Clone Development

Clones of these hybridomas are carefully selected based on a number of criteria including robust growth and efficient production of a single clone of antibody that is specific to the intended target. The best clones move on to applications testing. The steps include:
 

• Immunogen design and construction
• Immunization of host animal
• Hybridoma creation
• ELISA or application-specific screening of antibodies from clones
• Application testing, including WB, ELISA, ChIP, IF, IHC, or biofunctional assays

Multiple Application Validation

Antibody clones are then tested in a variety of assays to see which applications they are suited for. As an example, clone 13A3-1 for phosphorylated STAT3 (Tyr705) demonstrated excellent performance in flow cytometry, western blot, and chromatin immunoprecipitation.

Thus, the clone cross-validates itself by demonstrating functionality across orthogonal testing methods. Additionally, the biological induction of the phosphorylated state using IL-6 further validates the specificity of the antibody.

Gene Knockdown Testing

Validation of our antibodies in knockout (KO) or knockdown (KD) models demonstrate the specificity and quality of our products. When validating our antibodies in KO/KD, we often utilize cells which have been modified by CRISPR/Cas9 technology or siRNA-mediated silencing. We have partnered with Horizon Discovery, part of Revvity, to generate the KO/KD models using their state-of-the-art gene silencing reagents and cell lines. For more information, please visit the website. To see a complete list of our KO/KD-validated antibodies, click here. Visit our Knockout and Knockdown Validation page for more information on how we qualify our antibodies for specificity and performance.

(Left) HEK293 cells were transfected with non-targeting siRNA (positive control, Horizon Cat. No. D-001810-10) or with BAK1 siRNA (low expression control, Horizon Cat. No. L-003305-00) using DharmaFECT 1 Transfection Reagent (Horizon Cat.No. T-2001). Cells were fixed with 4% PFA Fixation Buffer (Cat. No. 420801), permeabilized with ice cold methanol and then blocked with 5% FBS. Cells were stained with Purified anti-BAK1 (clone W23097D) (right panels) or Rat IgG2a, κ isotype control (Cat. No. 400502) (left panels) followed by Alexa Fluor® 647 Goat anti-rat (Cat. No. 405416). Nuclei were counterstained with DAPI (Cat. No. 422801). The images were captured on a Revvity Operetta CLS™ High Content Analysis System with a 63X objective. Scale bar: 50 μm.