Knockout Validated Antibodies

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.

Solution

Validating an antibody's specificity is crucial to ensuring the absence of nonspecific binding. Removal of target protein expression within a given cell or tissue type generated by a gene KO technique is the accepted standard for validation of specificity by testing against a true negative sample. The approach to address this is through knockout or knockdown of the target protein by CRISPR- or RNAi-mediated technologies. Antibody specificity is then qualitatively assessed by the absence of off-target binding in the KO control lysate or cell lines.

How do we validate our Knockout Antibodies?

We have created hundreds of CRISPR Knockout cell lines by introducing frame-shift mutations into the genomic DNA of target cells using sgRNA/CAS9.
The CRISPR knockout cells are lysed and Western Blotting is performed using Wild-Type (WT) cells as a positive control. A truly specific antibody recognizes the target protein of right molecular weight in the WT cell lysate, but not in the KO cell lysate. KO validation serves as a true negative control to confirm antibody specificity to the protein of interest.

Figure 1. Schematic representation of how KO validated antibodies are created using CRISPR technology.

Images

CABT-L2021

Knockout Verification

CABT-L2021
Lane 1: Wild-type RaW264. 7 cell lysate.
Lane 2: FADD knockout RaW264. 7 cell lysate. Predicted MW: 23kDa, Observed MW: 27kDa

CABT-L2065

Knockout Verification

CABT-L2065
Lane 1: Wild-type U87MG cell lysate. Lane 2: LTbR knockout U87MG cell lysate. Predicted MW: 45, 47kDa, Observed MW: 47kDa

CABT-L2088

Knockout Verification

CABT-L2088
Lane 1: Wild-type Jurkat cell lysate.
Lane 2: CASP2 knockout Jurkat cell lysate. Predicted MW: 10, 35, 51kd, Observed MW: 51kd

CABT-L2183

Knockout Verification

CABT-L2183
Lane 1: Wild-type Hela cell lysate.
Lane 2: SKP1 knockout Hela cell lysate. Predicted MW: 18, 19kd, Observed MW: 20kd

CABT-L2160

Knockout Verification

CABT-L2160
Lane 1: Wild-type 293T cell lysate.
Lane 2: SYK knockout 293T cell lysate. Predicted MW: 69, 72kd, Observed MW: 69kd

Western Blot CABT-L2160

Lane1: Mouse Spleen Tissue;
Lane2: Rat Spinal Cord Tissue.

At Creative Diagnostics, we use an extensive library of KO cell lines to validate our antibodies. Our KO cell lines are generated via CRISPR-Cas9 and editing is verified by Sanger sequencing or NGS. We also employ proteomic validation, where possible.

Creative Diagnostics is committed to providing the best antibodies available. Antibodies that have passed the KO test will be labeled with the 'Knockout Validated' icon.

See our complete list of Knockout Validated antibodies

Primary Antibodies