Utilizing two independent antibodies for the same protein target can be a useful tool when testing for antibody specificity. In the ideal scenario, two antibodies are used that target nonoverlapping epitopes of an antigen. By obtaining comparable results from antibodies that recognize independent regions of the same target protein, this allows for increased confidence that these antibodies are specific and suitable for the detection of their intended target. Independent antibody testing is one strategy we use to validate Invitrogen antibodies for research use.

Independent validated antibodies

Common applications of independent antibody validation would be obtaining similar detection patterns in multi-lysate western blots, IHC arrays, immunofluorescence of multiple cell lines, immunoprecipitation, flow cytometry, and other antibody applications.

The use of independent antibodies is theoretically straightforward, however in reality hard to predict. Results can vary from sample to sample depending on sample prep, buffer systems, orientation in a multi-protein complex and other parameters that can influence protein conformation. Epitope accessibility can therefore vary considerably by one or both of the tested antibodies. To counter these complications, we also use a polyclonal-monoclonal approach with antibodies against the same target to verify antibody specificity. Both, the monoclonal and the polyclonal pool are expected to show similar detection patterns, despite the expected differences in sensitivity.

Independent antibody validation data

The following examples demonstrate how we use independent antibodies to verify target specificity.

In the first example (Figure 1), western blot analysis was performed on whole cell extracts of SKOV3, MDA-MB-231, MCF7, GTL16, HCT116, PC3, A431, PANC-1 and A549 with three independent VCP antibodies. Antibody specificity was demonstrated by showing that antibodies raised against the same target protein perform similarly. VCP Polyclonal Antibodies (Cat. No. PA5-27323, PA5-29638, and, PA5-22257) all tested in parallel shows similar expression of VCP in the cell lines tested.

Figure 1. Western blot analysis was performed on whole cell extracts. 30 µg lysate of SKOV3 (Lane 1), MDA-MB-231 (Lane 2), MCF7 (Lane 3), GTL16 (Lane 4), HCT116 (Lane 5), PC3 (Lane 6), A431 (Lane 7), PANC-1 (Lane 8), and A549 (Lane 9). The blot was probed with VCP Polyclonal Antibodies (Cat. No. PA5-27323, 1:8000 dilution) (left), (Cat. No. PA5-29638, 1:8000 dilution) (center), (Cat. No. PA5-22257, 1:8000 dilution) (right) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy chain) Superclonal Secondary Antibody, HRP (Cat. No. A27036, 0.25 µg/ml, 1:4000 dilution). A 97 kDa band corresponding to VCP was observed in all cell lines. Known quantity of protein samples were electrophoresed using NuPAGE 4-12 % Bis-Tris Gel (Cat. No. NP0321BOX), XCell SureLock Mini-Cell and XCell II Blot Module (Cat. No. EI0002), and Novex Sharp Pre-Stained Protein Standard (Cat. No. LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with the iBlot 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Cat. No. 32106).

In another example (Figure 2), antibody specificity was demonstrated by showing that antibodies raised against the same target protein ALDH2, perform similarly. Immunofluorescence of ALDH2 using ALDH2 Polyclonal Antibody (Cat. No. PA5-11483) performed along with another ALDH2 antibody (Cat. No. PA5-29717) shows similar localization pattern in Hep G2 cells.

Independent validation of ALDH2 antibodies used in immunofluorescence

Figure 2. Immunofluorescence analysis of ALDH2 was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. Panels a and b were labeled with ALDH2 Polyclonal antibody (Cat. No. PA5-11483) at 1:50 dilution and panels c and d were labeled with ALDH2 Polyclonal antibody (Cat. No. PA5-29717) at 1:100 dilution. After an overnight incubation at 4 °C, the cells where then labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034) at a dilution of 1:2,000 for 45 minutes at room temperature.

Verifying target specificity of Invitrogen antibodies using independent antibody verification

Invitrogen antibodies that have been verified using independent antibodies 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.

Advanced Verification

Thermo Fisher Scientific is committed to adopting higher validation standards for the Invitrogen antibody portfolio. We have implemented additional specificity tests to help ensure the highest confidence levels in our products. You can identify the products that have already undergone this testing with the Advanced Verification badge, shown above. This badge can be found in antibody search results and at the top of product webpages. The data supporting the Advanced Verification status can be found in the product specific data galleries. To learn more about our testing standards, please visit Invitrogen Antibody Validation.

*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.

For Research Use Only. Not for use in diagnostic procedures.