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Antibodies are amongst the most commonly used tools in life science and clinical research for the study of proteins and their functions within various biological pathways and diseases.
A good antibody exhibits target specificity 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 are specific in this way, with many showing cross-reactivities with off-target proteins. This has led to the recognized issue of experimental irreproducibility.1 These 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 that does not express the target protein.4 A specific antibody should yield no signal when tested in a KO cell line while giving the specific target protein signal in the wild-type (WT) 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 haploid cell lines to perform antibody validation. These KO cell lines are generated via CRISPR-Cas9 and certified via Sanger sequencing. 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.6-8
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. 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
Figure 2. Cdk6 Mouse monoclonal product (ab54576). The expected band at the correct MW disappears in the KO sample; thus, the antibody recognizes its intended target. However, extra bands are also present in both WT and KO samples. This may be due to antibody cross-reactivity with other protein 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 antibody – weak signal in the KO sample
Figure 3. Akt1 RabMAb® product (ab32038). The expected band at the correct MW is present in the KO sample but at a lower intensity compared to WT sample. However, the antibody may be exhibiting cross-reactivity with other protein family members/isoforms which are present at the same molecular weight in the sample 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
Figure 4. Akt1 rabbit polyclonal product (ab91505). The expected MW band is still present in the KO at the same intensity as the WT. Use of a bench-mark antibody shows a target band in the WT and no band in KO samples.
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 antibody – no target band in both KO and WT samples
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.
See our complete list of KO-validated antibodies.
The application of CRISPR-Cas9 technology has allowed us to develop an extensive range of KO cell lines and lysates, so that we can provide you with reliable antibodies, saving you time and money. Each KO cell line is individually cloned, validated by Sanger sequencing, and supplied with the parental WT control lysate to allow the biological impact of the knock-out to be assessed within a consistent cellular background.
See our complete list of knockout cell lysates
See our complete list of knockout cell lines