Knockout and Knockdown Antibody Validation*

Validating* an antibody’s specificity is crucial to ensuring the absence of non-specific binding and, therefore, the highest level of functionality. Testing antibody performance against genetically modified samples is one way to verify that an antibody recognizes a specific target. This can be done using a variety of methods, including mouse knockout models, dominant negative mutants, morpholinos, siRNA, and most recently, gene editing. Thermo Fisher Scientific is committed to providing the best antibodies available. We are currently re-testing the entire portfolio of Invitrogen antibodies to verify that they are specific to the indicated targets.

Knockout / knockdown validated antibodies

Utilizing the CRISPR-Cas9 system, scientists can create knockout cell models that can be subsequently used as robust controls for validating antibody specificity. The CRISPR-Cas9 system employs a noncoding single guide RNA (sgRNA) molecule to "guide" the CRISPR-associated Cas9 endonuclease to its intended target gene, where it cleaves the DNA. This DNA cleavage results in target gene knockout. In this way, CRISPR-Cas9 technology is used to ablate the target protein’s expression in appropriate cell models, thus making it a suitable negative control for verifying antibody specificity (Figure 1).

CRISPR-Cas9 can also be used to simultaneously test antibody specificity for multiple signaling proteins in a pathway by knocking out expression of upstream mediators. This multiplexing capability allows for streamlined and high-throughput antibody validation. Considering this advantage, CRISPR-Cas9 technology becomes the preferred solution for validating antibodies by genetic modification.

Figure 1. Schematic of CRISPR-Cas9 knockout–mediated validation of antibody specificity.

In the example below, antibody specificity was demonstrated by CRISPR-Cas9 mediated knockout of target protein. Loss of signal was observed for target protein in ErbB2 (HER-2) knockout (KO) cell line using ErbB2 (HER-2) Monoclonal Antibody (6C2) (Cat. No. MA5-15702).

In the example below, immunofluorescence analysis of EGFR was done on 70% confluent log phase A431 cells. Antibody specificity was demonstrated by CRISPR-Cas9 mediated knockout of target protein. Loss of EGFR expression was observed in EGFR knock out cell line as compared to the control cell line using EGFR Monoclonal Antibody (H11) (Cat. No. MA1-12693).

Figure 4. CRISPR-Cas9 experimental workflow for demonstrating antibody specificity.

RNA interference (RNAi) technology takes advantage of a cell’s natural machinery to effectively knock down expression of a gene of interest. It is a widely used method to validate antibody specificity.

In mammalian cells, short pieces of double-stranded RNA, otherwise known as short interfering RNA (siRNA), initiate the degradation or knockdown of a specific, targeted cellular mRNA. In this process, the antisense strand of the siRNA duplex becomes part of a multi-protein complex called the RNA-induced silencing complex (RISC). RISC then identifies the complementary mRNA and cleaves it at a specific site. Next, this cleaved message is targeted for degradation, ultimately resulting in the loss of protein expression.

There are different methods for performing RNAi experiments. RNAi can be achieved by transfecting target cells with a pool of synthetic small RNAs or a pool of siRNA obtained via in vitro cleavage (in vitro dicing of target RNA). RNAi can also be achieved by transfecting cells with short hairpin RNA (shRNA) vectors. shRNA is processed within the cell, and the in vivo-generated siRNA can then target its specific mRNA molecule for degradation. Nontargeting controls can be used to test for specificity of the knockdown.

Figure 5. Principle of siRNA-mediated knockdown of target mRNA. The figure shows the use of in vitro-synthesized siRNA in a typical RNAi experiment.

In the example below, Invitrogen Silencer Select siRNAs are transfected into cells in a 96-well format, followed by RT-qPCR. The most efficient knockdown (based on cell type and siRNA transfection conditions) is identified through gene expression analysis. The chosen screen is then scaled up for validation of antibody specificity through western blotting and immunocytochemistry (ICC).

Figure 6. Schematic of the knockdown strategy used to validate antibody specificity.

Invitrogen antibodies that have been verified against genetically modified samples to bind their target are indicated with a “verified specificity” symbol in search results and on relevant product pages. The data showing the verification will be provided on each product page.

*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.