Knockout validation antibodies

Knockout (KO) validation: confirming antibody specificity

To ensure you have highly specific antibodies, we use KO validation via CRISPR/Cas9 genome-editing to give you the reliable results your research demands.

The need

Antibodies are amongst the most commonly used tools in life science and clinical research to study proteins and their functions within various biological pathways and diseases. With their specificity and sensitivity, antibodies help to identify proteins of interest at different expression levels. An increasing number of studies have shown that not all antibodies are specific, leading to a growing issue around experimental irreproducibility. This reproducibility issue is in part due to the cross-reactivity and off-target protein binding. These unspecific antibodies result in wasted resources and compromise the advancement of science (Bradbury & Plückthun, 2015; Baker, 2015; Mullane et al., 2015).

The solution: knockout validation

One of the most accepted and trusted validation processes for antibody specificity is knockout (KO) validation (Madhusoodanan, 2014; Rhodes & Trimmers, 2008). KO validation is an incredibly robust technique to confirm specificity by testing the antibody of interest in a KO cell line or tissue that does not express the target protein.

If you compare KO cell line data against a that from a wild-type (WT) cell line, a specific antibody should produce no signal the KO cell line but give a specific signal in the WT cell line. In this way, KO validation serves as a true negative control to confirm antibody specificity to the protein of interest (Bordeaux, et al., 2010).

We use the extensive library of human KO haploid cell lines generated via CRISPR/Cas9, available from Horizon Discovery. This type of KO cell line provides a complete loss-of-function phenotype from a single allele KO and eliminates any masking of the knockout from a second allele seen in diploid cell models (Bürckstümmer T et al. 2013; Reiling JH et al. 2011; Carette JE et al. 2009)

Knockout validation testing results

When we KO validate our antibodies, we primarily use western blot to assess the results. Here are the different types of western blot outcomes from our antibody testing and how we deal with each of them.

Specific

  • Result: expected molecular weight (MW) band disappears in the KO sample.
  • Next steps: KO-based specific data added to the datasheet. Antibody guaranteed for specificity via KO testing. 


Figure 1. Cdk4 RabMAb® product – ab108357. The expected band at the correct MW disappears in the KO sample.

All lanes: Anti-Cdk4 rabbit monoclonal antibody [EPR4513] (ab108357) at 1/1000 dilution 

Lane 1: Wild-type HAP1 cell lysate
Lane 2: Cdk4 Knockout HAP1 cell lysate

Predicted band size: 34 kDa
Observed band size: 34 kDa

Loading control (red band):  Anti-beta Actin antibody (ab8226) 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 1/10,000 dilution

Partially specific – ​extra bands

  • Result: expected MW band disappears in KO, but extra bands are present at a distinct MW.
  • Next steps: we add KO-based specific data to the datasheet. Additional research and testing will be performed to identify extra bands.

Figure 2. Cdk6 Mouse monoclonal product – ab54576. The expected band at the correct MW disappears in the KO sample, therefore it recognizes its intended target. However, extra bands are also present in both WT and KO samples. This may be due to it cross-reacting with other family members/isoforms or unrelated proteins.

All lanes:  Anti-Cdk6 antibody (ab54576) at 1/1000 dilution

Lane 1: Wild-type HAP1 cell lysate
Lane 2: Cdk6 Knockout HAP1 cell lysate

Predicted band size: 37 kDa
Observed band size: 37 kDa

Loading control (red band):  Anti-beta Actin antibody (ab8227) at 1/1000 dilution

Secondary antibodies:
IR Dye 800 Goat anti-Mouse IgG (H + L)
IR Dye 680 Goat anti-Rabbit IgG (H + L)
both at 1/10,000 dilution

Non-specific – ​weak signal in the KO sample

  • Result: expected MW band still present in KO at a lower intensity compared to WT. A benchmark antibody resolves a target band in WT and no band in KO. 
  • Next steps: we add KO-based specific data to the datasheet. Additional research and testing will be performed to identify the reasons behind the signal in the KO sample.

Figure 3. Akt1 RabMAb® product – ab32038. The expected band at the correct molecular weight is present in the KO sample but at a lower intensity compared to WT sample. However, it might cross-react with other family members/isoforms which are present at the same molecular weight as the target protein.

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 (red band):  Anti-beta Actin antibody (ab8226) 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 1/10,000 dilution

Non-specific – signal in the KO sample

  • Result: expected MW band still present in KO at the same intensity as WT. A bench-mark antibody resolves a target band in WT and no band in KO.
  • Next steps: we remove the antibody from the catalog, and we recommend an alternative antibody with confirmed specificity.

Figure 4. Akt1 rabbit polyclonal product – ab91505. The expected MW band still present in the KO at the same intensity as the WT. Bench-marker shows a target band in WT and no band in KO. 

All lanes:  Anti-Akt1 rabbit polyclonal antibody (ab91505) at 1:1000

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 (red band):  Anti-beta Actin antibody (ab8226) 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 1/10,000 dilution

Non-specific – no target band in both KO and WT samples

  • Result: does not recognize the target band in both WT and KO samples. A bench-mark antibody resolves a target band in WT and no band in KO. 
  • Next steps: we remove the antibody from the catalog, and we recommend an alternative antibody with confirmed specificity.

Knockout-validated antibodies: our promise

When you see the knockout 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.

See our complete list of KO-validated antibodies.




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