Peptide array is a powerful tool for identifying protein-protein interactions where antibodies are allowed to independently interact with the target peptide and a collection of closely related peptides. Afterward, antibody specificity is evaluated using chemiluminescent detection. Peptide array testing is one strategy we use to validate Invitrogen primary and secondary antibodies for research use. A strong binding signal to the intended peptide(s) and no cross reactivity demonstrates that the antibody is specific to the target of interest.

Peptide array validated antibodies

Peptide array validation data

Peptide array is performed to validate the specificity of antibodies used to study epigenetics, such as post-translational modifications. In the example below, antibody specificity was determined for histone modification by this method. Histone peptides containing 384 different modification combinations spotted in duplicate on an array are used to verify antibody specificity. In a typical experiment, the array is probed with a modification specific antibody followed by a HRP-labeled secondary antibody and chemiluminescent detection. In the example below (Figure 1) a methyl-histone H3 (Lys4) recombinant polyclonal antibody (Cat. No. 710795) was used to specifically detect histone H3 when methylated on lysine 4. The data show minor cross-reactivity to dimethylated lysine H3 and methylated histone lysine 20. The antibody did not detect other modifications of histone H3 or other histones and their modifications.

Figure 1. Peptide arrays performed using a recombinant polyclonal antibody (Cat. No. 710795) and two other suppliers’ antibodies targeting H3K4me1. Arrays were incubated overnight with a 1:2,000 dilution of primary antibody. After washing, the arrays were incubated with Goat anti–Rabbit IgG (Heavy chain) Superclonal Recombinant Secondary Antibody (Cat. No. A27033) at a dilution of 1:5,000 for one hour. Arrays were incubated with SuperSignal West Pico substrate (Cat. No. 34578) and then visualized using a myECL Imager.

Peptide array can be utilized to provide an assessment of the domain and subclass specificity of Invitrogen secondary antibodies. Cross-reactivity is assessed by adding a collection of taxonomically proximal immunoglobulins, crystallizable fragment (Fc) regions, or individual heavy and light chains to a solid surface. ELISA analysis is run to ensure the specific of the secondary antibody – as in the case of Goat anti-Human IgG (Kappa light chain) Recombinant Secondary Antibody, HRP (Cat. No. A56864).

In the example below, a western blot analysis of kappa and lambda specific whole IgG, antigen binding fragment (Fc), whole IgG, and crystallizable fragment (Fc) controls was performed. In addition, kappa and lambda specific whole IgG, Human IgG crystallizable fragment (Fc), and an array of immunoglobulins from various species were quantified via ELISA. In both cases, the Goat anti-Human IgG (Kappa light chain) Recombinant Secondary Antibody, HRP was used to specifically detect the kappa light chain. The data also shows no reactivity with the lambda light chain, human IgG Fc, or immunoglobulins from various other species.

Western blot and bar graph showing protein quantification via ELISA demonstrating the specificity of Goat anti-Human IgG Recombinant Secondary Antibody

Figure 2. Peptide array-based verification of a recombinant secondary antibody (Cat. No. A56864). A 1 µg of indicated samples were run on a Novex NuPAGE 4-12%, Bis-Tris gel, transferred onto a nitrocellulose membrane and probed with Goat anti-Human IgG (Kappa light chain) Recombinant Secondary Antibody, HRP (Cat. No. A56864) at a dilution of 1:5000 and detected on an iBright FL1500. B. Indicated samples were coated at a concentration of 25 ng/well on a 96-well plate and probed with Goat anti-Human IgG (Kappa light chain) Recombinant Secondary Antibody, HRP (Cat. No. A56864) at a dilution of 1:2000 and detected on a Thermo Scientific™ Varioskan™ LUX multimode microplate reader.

Verifying target specificity of Invitrogen antibodies using peptide arrays

Invitrogen antibodies that have been verified with peptide arrays 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 high 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.

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