Genetic Knockout (KO) Antibody Validation and the Reproducibility Crisis


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

Experiment reproducibility 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.

All 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’s 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. Search our KO validated antibodies and KO cell lysates (293T and HeLa) to view validation data, or request a KO cell line.

Human K562 Vimentin KO Antibody Validation, ICCVimentin was detected in immersion fixed K562 human chronic myelogenous leukemia cell line but is not detected in Vimentin knockout (KO) K562 Human Cell Line cell line using Goat Anti-Human Vimentin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2105) at 5 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 493-conjugated Anti-Goat IgG Secondary Antibody (green; Catalog # NL003) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

 

Why KO/Knockdown 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, Scientists may circumvent the resource intensive CRISPR/Cas9 KO production protocol by ordering custom KO cell lines and cell lysates. 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.

Human HeLa p62/SQSTM1 KO Antibody Validation, ICCp62/SQSTM1 was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line but is not detected in p62/SQSTM1 knockout (KO) HeLa cell line using Mouse Anti-Human/Mouse/Rat p62/SQSTM1 Monoclonal Antibody (Catalog # MAB8028) at 3 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

 

Novus Biologicals Guaranteed KO Validated Antibodies

To eliminate unreliability, Bio-Techne brands Novus Biologicals and R&D Systems are joining global initiatives dedicated to standardizing antibody validation. In alignment with this mission, Novus Biologicals provides KO validated antibodies, custom KO cell lines and over 1,750 KO cell lysates. In partnership with gene-editing firms EdiGENE Corp. and B-MoGen Biotechnologies Inc., biologically relevant data is being produced for 730 antibody products, and validation has been confirmed for over 90 targets and counting. In accordance with recommendations from the International Working Group for Antibody Validation (IWGAV) and the Global Biological Standards Institute (GBSI), Novus Biologicals utilizes Five Pillars of Antibody Validation to ensure antibody specificity under different conditions and applications. As such, Novus Biologicals guarantees every product will work in the application and species listed on our website and datasheets.

Human HeLa YAP1 KO, YAP1 Antibody Validation, WBKnockout Validated: YAP1 Antibody [NB110-58358] - Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and YAP1 knockout (KO) HeLa cell line. PVDF membrane was probed with 1:1000 of Rabbit Anti-Human YAP1 Polyclonal Antibody (Catalog # NB110-58358) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). Specific band was detected for YAP1 at approximately 75 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. This experiment was conducted under reducing conditions.

 

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