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Antibodies are some of the most widely used tools in life science and clinical research to study proteins and their functions within biological pathways and diseases.
A good antibody exhibits target specificity, selectivity, and sensitivity, allowing it to identify the protein of interest even at low expression levels. However, an increasing number of studies have shown that not all antibodies have target specificity, with many showing cross-reactivity with off-target proteins. This is an issue of experimental irreproducibility1 as non-specific antibodies result in wasted resources and compromise the advancement of science.2,3
Knock-out (KO) validation is a robust technique used to confirm antibody specificity by testing the antibody of interest in a KO cell line or tissue in which the target gene has been edited so that the target protein is not expressed.4 A specific antibody will detect the specific target protein signal in the unedited, wild-type (WT) cell line and yield no signal when tested in a KO cell line. In this way, KO validation serves as a true negative control to confirm antibody specificity to the protein of interest.4,5
At Abcam, we use an extensive library of human 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.
See our complete list of KO-validated antibodies
When KO-validating our antibodies, we primarily use western blot to assess the results. A range of western blot outcomes can result from KO validation, and the following examples demonstrate how we deal with each situation at Abcam.
Figure 1. Western blot of WT and KO samples probed with anti-EGFR antibody [E235] (ab32077) at 1/1000 dilution. The band of interest is present in the WT and absent in the KO sample.
All lanes: Anti-EGFR antibody [E235] (ab32077) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate
Lane 2: EGFR knockout A549 cell lysate (ab286394)
Lane 3: Wild-type HeLa cell lysate
Lane 4: EGFR knockout HeLa cell lysate (ab255385)
Predicted band size: 134 kDa
Observed band size: 160 kDa
Loading control (red band): Mouse anti-Alpha Tubulin [DM1A] (ab7291) at 1/20000
Secondary antibodies:
IR Dye 800 Goat anti-Rabbit IgG (H + L)
IR Dye 680 Goat anti-Mouse IgG (H + L)
both at 1/10,000 dilution
Figure 2. Western blots of WT and KO samples probed with Anti-FANCA/FAA antibody (97578) at 1/1000. The band of interest at 163 kDa is present in the WT and absent in the KO sample in both blots, so the antibodies specifically bind to Rab3a. However, ab3335 shows several other bands present in both WT and KO, showing it is not selective. We recommend ab201457 as it is more selective, with only one faint non-specific band detected at 65 kDa.
Lane 1: Wild-type SK-N-FI cell lysate
Lane 2: RAB3A knockout SK-N-FI cell lysate (ab288708)
Lane 3: Human brain cell lysate
Lane 4: HEK-293 cell lysate
Predicted band size: 24 kDa
Observed band size: 27 kDa
Loading control (magenta band): Mouse anti-Alpha Tubulin [DM1A] (ab7291) at 1/20000 dilution
Secondary antibodies: IR Dye 800 Goat anti-Rabbit IgG (H + L)
IR Dye 680 Goat anti-Mouse IgG (H + L) both at 1/20,000 dilution
Cross-reactive antibody
Figure 3. Anti-Akt1 antibody tested on KO lysates. The expected band at the correct MW is present in the KO sample but at a lower intensity compared to WT sample. Additional overexpression testing showed that the antibody also detects AKT3 (data shown on the datasheet), which is the same molecular weight as AKT1. The antibody was renamed.
All lanes: Anti-Akt1 rabbit monoclonal antibody (ab32038) at 1/1000 dilution
Lane 1: Wild-type HAP1 cell lysate
Lane 2: Akt1 Knockout HAP1 cell lysate
Predicted band size: 55 kDa
Observed band size: 55 - 60 kDa
Loading control (magenta band): Mouse anti-GAPDH antibody [6C5] (ab8245) at 1/20,000 dilution at 1/1000 dilution
Secondary antibodies:
IR Dye 800 Goat anti-Rabbit IgG (H + L)
IR Dye 680 Goat anti-Mouse IgG (H + L)
both at 1/20,000 dilution
Figure 4. Western blot of WT and KO samples probed with Anti-TGF beta Receptor I antibody (ab31013) at 1/1000 dilution. A non-specific band is present in the WT and the KO sample.
Lane 1: Wild-type A549
Lane 2: TGFBR1 knockout A549 cell lysate (ab277894)
Lane 3: A431 cell lysate
Lane 4: MOLT-4 cell lysate
Predicted band size: 27 kDa
Observed band size: 35 kDa
Loading control (red band): Mouse anti-GAPDH antibody [6C5] (ab8245) at 1/20,000 dilution
Secondary antibodies:
IR Dye 800 Goat anti-Rabbit IgG (H + L)
IR Dye 680 Goat anti-Mouse IgG (H + L)
both at 1/20,000 dilution
When you see the KO validated seal, you can trust that the antibody has not only been validated in the recommended applications and species, but its specificity has been confirmed through our in-house KO validation approach. 3,600 of our recombinant antibodies are KO validated, offering unrivaled specificity.
See our complete list of KO-validated antibodies
The application of CRISPR-Cas9 technology paired with our high-throughput platform allows us to automate the production of our CRISPR knock-out cell lines at scale. This reduces the time typically required to produce gene-edited monoclonal cell lines, so you can gain back valuable lab time, have confidence in scalable and long-term supply, and achieve consistent results over time. Our ready-made cell lines are available for delivery within five days, saving you valuable lab time.
Each KO cell line is individually cloned, validated by genomic sequencing and proteomic analysis in many cases, and supplied with the parental WT control to allow the biological impact of the knock-out to be assessed within a consistent cellular background.
See our complete list of knock-out cell lysates
See our complete list of knock-out cell lines