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.

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, whole cell lysates and nuclear extracts from ER alpha–positive MCF7, T47D, SK-BR-3, and ER alpha–negative Hs578T cells were analyzed with two independent ER alpha antibodies, one a polyclonal antibody and one a monoclonal antibody. The immunogen for the polyclonal was a short synthetic peptide derived from the C-terminus of human ER alpha protein, while the immunogen for the monoclonal was a partial protein of 35 kDa stemming from the C-terminal region (aa 302–595) of human ER alpha expressed in E. coli. Differences in species reactivity, as well as mass spectrometry analysis, suggested that these antibodies have adequately different binding profiles to their target and are appropriate for specificity verification. As expected, ER alpha was detected as the prominent band, mostly in the nuclear extract of ER-positive cell lines. In addition, both antibodies showed the same pattern of ER alpha enrichment in the nuclear fractions, further supporting specificity of the antibodies.

Western blot analysis of Estrogen Receptor
Western blot analysis of Estrogen Receptor

Western blot analysis of Estrogen Receptor (ER). Electrophoresis was performed after loading 20 µg of whole cell lysates (WCL) or nuclear extracts (NE) from the indicated breast cancer cell lines, and 10 µL of PageRuler Prestained Protein Ladder (Cat. No. 26616) per well onto a Novex 4–20% Tris-glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane (Cat. No. 88518) and blocked with 5% milk in TBST for at least 1 hr at room temperature. ER was detected at ~43 kDa (top panel) using an ER polyclonal antibody (Cat. No. PA5-16440) at a dilution of 1:500 in blocking buffer overnight at 4°C on a rocking platform, followed by an HRP-conjugated goat anti–rabbit IgG secondary antibody (Cat. No. 31460) at a dilution of 1:40,000. Chemiluminescent detection was performed using SuperSignal West Dura substrate (Cat. No. 34075). To check for equivalent protein loading in each lane, the blot was probed with an HDAC1 polyclonal antibody (Cat. No. PA1-860, bottom panel) The molecular weight of the endogenous protein detected is consistent with isoforms ER alpha or ER beta.

Western blot analysis of Estrogen Receptor (ER). Electrophoresis was performed after loading 20 µg of whole cell lysates (WCL) or nuclear extracts (NE) from the indicated breast cancer cell lines, and 10 µL of PageRuler Prestained Protein Ladder (Cat. No. 26616) per well onto a Novex 4–20% Tris-glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane (Cat. No. 88518) and blocked with 5% milk in TBST for at least 1 hr at room temperature. ER was detected at ~48 kDa (top panel) using an ER monoclonal antibody (Cat. No. MA5-13065) at a dilution of 1:1,000 in blocking buffer overnight at 4°C on a rocking platform, followed by an HRP-conjugated goat anti–mouse IgG secondary antibody (Cat. No. 31430) at a dilution of 1:40,000. Chemiluminescent detection was performed using SuperSignal West Dura substrate (Cat. No. 34075). To check for equivalent protein loading in each lane, the blot was probed with an HDAC1 polyclonal antibody (Cat. No. PA1-860, bottom panel) The molecular weight of the endogenous protein detected is consistent with isoforms ER alpha or ER beta.

 


In another example, two independent monoclonal antibodies were used to analyze beta-catenin: mouse monoclonal beta-catenin antibody, clone 15B8, and mouse monoclonal beta-catenin antibody, clone 6F9. As shown below, both antibodies showed good correlation in the detection of human, mouse, and rat beta-catenin. Interestingly, clone 15B8 also detected beta-catenin in nonhuman primate COS7 cells, while clone 6F9 did not detect beta-catenin in the same lysate. The immunogen for both antibodies was recombinant human beta-catenin protein.

Western blot analysis of beta-Catenin
Western blot analysis of beta-Catenin.

Western blot analysis of beta-Catenin. Electrophoresis was performed after loading 50 µg of the indicated whole cell lysates onto a 4–20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hr. The membrane was probed with a beta-catenin monoclonal antibody (Cat. No. MA1-301) at a dilution of 1:1,000 overnight at 4°C on a rocking platform, washed in TBS/0.1% Tween 20, and probed with a goat anti–mouse IgG HRP secondary antibody (Cat. No. 31430) at a dilution of 1:20,000 for at least 1 hr. Chemiluminescent detection was performed using SuperSignal West Pico substrate (Cat. No. 34080). 

Western blot analysis of beta-Catenin. Electrophoresis was performed after loading 50 µg of the indicated whole cell lysates per well onto a 4–20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hr. The membrane was probed with a beta-catenin monoclonal antibody (Cat. No. MA1-300) at a dilution of 1:1,000 overnight at 4°C on a rocking platform, washed in TBS/0.1% Tween 20, and probed with a goat anti–mouse IgG HRP secondary antibody (Cat. No. 31430) at a dilution of 1:20,000 for at least 1 hr. Chemiluminescent detection was performed using SuperSignal West Pico substrate (Cat. No. 34080).

 


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.

*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.