Knockout (KO) Validated Antibodies and the Reproducibility Crisis

Reproducibility Initiative: Knockout (KO) and Knockdown (KD) Validation

The reproducibility of experiments depends on antibody specificity and is critical to producing consistent results. Antibody cross-reactivity (the off-target association of antibodies with proteins beyond the target of interest) produces ambiguous and inconsistent assays, and prevents scientists from reproducing their original results.

Our antibodies are produced following standard procedures to prevent cross-reactivity, but quality controls are still essential for verifying reagent specificity. Using the CRISPR/Cas9 system, the ‘genetic strategy’ of gene-knockout (KO) has emerged as an ideal tool for antibody-specificity validation. KO validation uses CRISPR guide RNA (gRNA) to direct the Cas9 endonuclease to the gene of interest through sequence-specific targeting, producing double-stranded breaks around targeted sites. These breaks induce repair mechanisms to produce a frameshift mutation, insertion or deletion, which may be used to produce a KO cell line. Antibody specificity is then qualitatively assessed by the absence of off-target binding in the KO control lysate or cell lines, as shown below in a K562 Vimentin KO. This process is used to produce KO cell lysates for independent antibody validation.

Why choosing KO over KD Validation?

Gene knockdown (KD) utilizes siRNA/shRNA to target mRNA for degradation, essentially preventing the translation of protein. This method reduces the expression of the encoded target protein, but is temporary (due to degradation) and unreliable because mRNAs may evade the siRNA/shRNA RNAi mechanisms. Unlike gene KD, gene KO employs CRISPR-Cas9 to remove the gene genome-wide. Whole-genome knockout produces a more stable negative control verifiable by ICC and Western Blot, among others. To validate antibodies independently, custom engineered KO cell lines can be ordered from Bio-Techne to circumvent the resource intensive CRISPR/Cas9 KO production protocol. In the KO validation below, a HeLa human cervical epithelial carcinoma cell line is compared with a successful p62/SQSTM1 KO negative control, illustrating a confirmed validation.

Guaranteed KO Validated Antibodies

To eliminate unreliability, Bio-Techne has joined global initiatives dedicated to standardizing antibody validation. In alignment with this mission, we provide KO validated antibodies and offer custom KO cell lines. Biologically relevant data is being produced for 845 antibody products, and validation has been confirmed for over 200 targets and counting. In accordance with recommendations from the International Working Group for Antibody Validation (IWGAV) and the Global Biological Standards Institute (GBSI), we follow the Five Pillars of Antibody Validation to ensure antibody specificity under different conditions and applications. As such, we guarantee that every product will work in the application and species listed on our website and datasheets.