Testing antibody performance against genetically modified samples

Validating* an antibody’s specificity is crucial to ensuring the absence of nonspecific 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 retesting the entire portfolio of Invitrogen antibodies to verify that they are specific to the indicated targets.

Advanced Verification

The Advanced Verification badge is applied to products that have passed application and specificity testing. Advanced Verification testing of Invitrogen antibodies continues to expand across the portfolio. An antibody lacking the badge should be seen as an antibody that has not yet undergone testing – not a reflection of the specificity of the product. This badge can be found in the search results and at the top of the product specific webpages. Data supporting the Advanced Verification badges can be found in product specific data galleries.

CRISPR-Cas9 in knockout cell models

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.

Schematic of CRISPR-Cas9 knockout–mediated validation of antibody specificity

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 Anti-ErbB2 (HER-2) Mouse Monoclonal Antibody (Cat. No. MA5-15702).

ErbB2 antibody showed specificity by CRISPR-Cas9 mediated knockout in western blot

Figure 2. Western blot analysis of ErbB2 (HER-2) (Fig. 2 a) was performed by loading 30 µg of SK-BR-3 Control (lane 1) and SK-BR-3 ErbB2 knockout (lane 2) whole cell extracts.ErbB2 (HER-2) was detected at 185 kDa using ErbB2 (HER-2) Mouse Monoclonal Antibody (Cat. No. MA5-15702, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody HRP conjugate (Cat. No. A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in the histogram (Fig. 2 b). Loss of signal in CRISPR mediated knockout (KO) confirms that antibody is specific to ErbB2 (HER-2).

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).

EGFR antibody showed specificity by CRISPR-Cas9 mediated knockout in immunofluorescence

Figure 3. Cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were subsequently labeled with EGFR Mouse Monoclonal Antibody (Cat. No. MA1-12693) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A28175) at a dilution of 1:2000 for 45 minutes at room temperature. Nuclei (Blue) were stained with SlowFade Gold Antifade Mountant DAPI (Cat. No. S36938). F-actin (Red) was stained with Rhodamine Phalloidin (Cat. No. R415, 1:300). EGFR signal was observed in control cell line (panels a-d) and not in the EGFR knockout (KO) cell line (panels f-i). Panels e and j represent the respective no primary controls.

Cas9 experimental workflow demonstrating antibody specificity

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

 

RNAi in knockdown strategies

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).

SiRNA transfection plan

Schematic of the knockdown strategy used to validate antibody specificity.

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

Knockdown validation of SMAD2 antibody demonstrated by western blot

Figure 7. Western blot for antibody validation. (A) Western blot showing knockdown of SMAD2 (lane 3) in HeLa whole cell lysates after transfection with SMAD2-targeting siRNA, using Invitrogen SMAD2 ABfinity Rabbit Monoclonal Antibody (Cat. No. 700048). The knockdown is shown along with untreated and scrambled RNA as controls in lanes 1 and 2, respectively. Detection of actin was used as a loading control. (B) Relative quantitation of the knockdown of SMAD2 bands on the western blot when compared to the untreated and scrambled siRNA as the positive control. The intensity of each band is normalized using the relative intensities of the actin bands.

Knockdown validation of CHD7 antibody demonstrated by immunocytochemistry

Figure 8. Immunocytochemistry for antibody validation: Knockdown of CHD7 was achieved by transfecting SH-SY5Y cells with CHD7 specific siRNA (Silencer select Cat. No. s31140, s529331). Immunofluorescence analysis was performed on untransfected SH-SY5Y cells (panel a, d), transfected with non-specific scrambled siRNA (panel b, e) and CHD7 specific siRNA (panel c, f). Cells were fixed, permeabilized, and labelled with CHD7 Polyclonal Antibody (Cat. No. PA5-72964, 1:100 dilution), followed by Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034, 1:2000). Nuclei (blue) were stained using ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962), and Rhodamine Phalloidin (Cat. No. R415, 1:300) was used for cytoskeletal F-actin (red) staining. Reduction of specific signal was observed upon siRNA mediated KD (panel c, f) confirming specificity of the antibody to CHD7.

Verifying target specificity of Invitrogen antibodies using genetically modified samples

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.