The Invitrogen ABfinity recombinant rabbit monoclonal antibody platform is especially powerful for generating antibodies against posttranslational modifications, such as phosphorylation-specific antibodies. The ABfinity process utilizes the natural diversity of the rabbit immune system, which tends to generate antibodies with higher affinity and specificity than those generated in mice. In addition, the biomanufacturing process identifies a single clone that recognizes the phosphorylation state of the target. An ABfinity antibody combines the robustness of a polyclonal antibody with the specificity of a monoclonal antibody. Additionally, since ABfinity antibodies are recombinant, the antibody performance is consistent from lot to lot, requiring no subsequent immunization of animals and no chance of clonal drift.
Catalog includes over 700 antibodies
There are now over 700 ABfinity recombinant rabbit antibodies, and more than 100 of these have been developed for specific phosphorylation states. In addition to the rabbit monoclonal antibodies, we produce ABfinity oligoclonal antibodies, which are recombinant polyclonal antibodies. These antibodies are designed to provide the diverse binding characteristics found in polyclonal antibodies as well as the lot-to-lot reproducibility that can only be found in recombinant antibodies
Rigorous sample screening and functional testing help to ensure final product quality
All ABfinity antibodies are subjected to several rounds of screening during production. This allows identification of clones that exhibit proven performance in the desired application. Finally, where possible, we test the antibody on activated targets to show the antibody’s specificity for a particular phosphorylation state.
The combination of the production method, in-process sample screening, and further functional testing makes ABfinity phosphospecific antibodies highly specific, highly sensitive tools for the investigation of key phosphorylation events within the cell.
Performance characteristics of phospho–4E-BP1 (Thr46) antibody. Western blot analysis of 4E-BP1 [pT46] was performed on 30 µg each of protein in cell lysates from the following samples: lane 1, HEK 293; lane 2, HEK 293 treated for 10 min with 100 nmol/mL of EGF; lane 3, MCF7; lane 4, MCF7 treated for 15 min with 150 µg/mL of insulin. The Invitrogen NuPAGE 4–12% Bis-Tris gel, XCell SureLock Electrophoresis System, Sharp Prestained Protein Standard, and iBlot Dry Blotting System were used for the protein electrophoresis and blotting steps. Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hr at room temperature. 4E-BP1 [pT46] was detected at ~20 kDa using ABfinity 4E-BP1 [pT46] Recombinant Rabbit Oligoclonal Antibody at 1–2 µg/mL in 2.5% skim milk at 4°C overnight on a rocking platform. Goat Anti–Rabbit IgG HRP Secondary Antibody at 1:5,000 dilution was used, and chemiluminescent detection was performed using the Novex ECL Chemiluminescent Substrate Reagent Kit.
Phosphospecific flow cytometry antibody functional testing
Each of our phosphospecific flow cytometry antibodies and associated buffer systems are subjected to a variety of functional tests. Our goal is to provide scientists with the confidence that our antibodies will perform as expected in all their flow cytometry experiments.
Functional testing includes:
- Pathway-specific tests—verify a panel of cells which have had different pathways induced. Phosphospecific staining should only be seen in cells where the pathways have been activated.
- Cell type–specific tests—verify that phosphospecific staining is only seen in cell types where the protein of interest is expressed.
- Application testing—can include western blot, ELISA, and immunochemistry to help ensure the antibody performs as expected.
- Buffer compatibility tests—each phosphospecific antibody is tested in 3 different buffer systems to determine which works best with the antibody.
- Cross-reactivity testing—each phosphospecific antibody is tested in human and mouse cells to determine species cross-reactivity.
- Competitive performance—compare our antibodies with our competitors’ to help ensure our product performs similarly or better.
Pathway-specific testing with eBioscience and competitor clones. U937 cells treated as indicated and subjected to intracellular staining with anti–phospho STAT 6 antibody, APC. Red spectra are for untreated cells, blue spectra are for treated cells.
The International Working Group on Antibody Validation suggests guidelines for demonstrating antibody specificity
The International Working Group on Antibody Validation (IWGAV) published a set of guidelines for demonstrating the specificity of antibodies:
A proposal for validation of antibodies, Mathias Uhlen, Anita Bandrowski, Steven Carr, Aled Edwards, Jan Ellenberg, Emma Lundberg, David L Rimm, Henry Rodriguez, Tara Hiltke, Michael Snyder & Tadashi Yamamoto, Nature Methods (2016) doi:10.1038/nmeth.3995.
Unlike application testing, which shows that the antibody works in a specific application, functional testing shows that the antibody truly binds to the intended target.
The paper describes several “pillars”, each showing techniques that verify that the antibody binds to the intended target. The immunoprecipitation–mass spectrometry pillar (IP-MS) is unique as a testing technique in that the readout displays actual peptide sequences specific to the proteins captured in the immunoprecipitation. (See IP-MS article here). Genetic modification, through knockout and knockdown technologies, demonstrates specificity through elimination of the specific protein by decreasing the amount of a specific gene. The multiple-epitope method utilizes antibodies to different epitopes on the same target to demonstrate specificity of both antibodies.
The IWGAV is an independent group of international scientists with diverse research interests in the field of protein biology. The IWGAV is the first initiative of its size and scope to work toward strategic recommendations for antibody functional testing for both antibody producers and users. Thermo Fisher Scientific, a world leader in serving science, provided financial support to the IWGAV in 2015 to spearhead the development of industry standards and help combat the common challenges associated with antibody specificity and reproducibility.
Learn more about IP-MS and knockout/knockdown functional testing.
Aligning Invitrogen antibody functional testing with IWGAV guidelines
Research antibodies are critical for innovation and discovery, but the use of poorly characterized reagents can negatively impact the rigor and reproducibility of biomedical science. The International Working Group on Antibody Validation is tackling this issue by providing guidelines for antibody functional testing. Following these guidelines, Thermo Fisher Scientific is striving to redefine the criteria of antibody performance by testing the specificity of Invitrogen antibodies in its portfolio in accordance with the newly proposed conceptual pillars for antibody functional testing, and providing extensive application functional testing data for our antibodies. Initiatives have already been launched to test antibodies by IP-mass spectrometry, CRISPR, and siRNA in addition to biological treatments, epitope mapping, and antibody sequencing as dictated by the protein target, as well as providing extensive application functional testing on multiple platforms. On our product detail pages, you will begin to see more extensive data for both target specificity and applications, to give you confidence in the performance of Invitrogen antibodies in your intended experimental applications.
See an example of an extensively functionally tested antibody
To search our entire portfolio of Invitrogen antibodies, visit thermofisher.com/antibodies