Antibody Insider—Issue 2017 Q1
ABfinity Recombinant Rabbit Monoclonals—advantages for detection of phosphorylation
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.
Learn more about ABfinity antibodies
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.
Representative phospho flow cytometry antibody functional testing data
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.
For more information see Phospho Flow Cytometry Antibody Validation
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.
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
Super Bright polymer dyes—bright dyes for the violet laser
Invitrogen eBioscience Super Bright dyes are a line of bright fluorochromes, based on a polymer and its tandems, that are excited by the violet laser (405 nm). All Super Bright formats are named for their emission wavelength. These dyes are optimized for use in flow cytometry, and their brightness may allow for better discrimination of dim populations. Certain dyes exhibit decreased spillover into other violet channels or display less nonspecific interaction with other polymer dyes as compared to similar competitor reagents.
Fully compatible with other commonly used fluorescent molecules, formaldehyde- and methanol-based fixatives, and Invitrogen UltraComp eBeads microspheres, the Super Bright conjugates allow optimized flow cytometry multicolor antibody panel design.
The Super Bright dyes include:
- Super Bright 436—has an excitation maximum of 414 nm and an emission peak of 436 nm. This dye offers improved brightness over other non-polymer dyes used in the same channel, and is an alternative for Brilliant Violet 421 with similar resolution of positive and negative populations.
- Super Bright 600—a tandem dye consisting of Super Bright 436 and an acceptor dye that emits at 600 nm. This tandem polymer dye is comparable in brightness to Brilliant Violet 605.
Increase your options for multicolor panel design
Super Bright dyes can be used like traditional fluorophores in flow cytometry applications. However, if two or more Super Bright conjugated antibodies are combined in the same panel, the use of Super Bright Staining Buffer is recommended to minimize any nonspecific interaction that may occur between these polymer-based dyes. Super Bright Staining Buffer is formulated to be used at 5 µL per test, making it convenient for use in master mixes and cocktails.
Super Bright dyes increase your options for multicolor panel design and allow you to expand the utility of your violet laser. Super Bright antibody conjugates are the user-friendly alternative you have been waiting for.
Learn more about Super Bright dyes
10-color T cell subsetting panel. Human peripheral blood cells were aliquoted in Super Bright Staining Buffer, then surface-stained with the indicated reagents. Samples were then fixed and permeabilized according to the Foxp3/Transcription Factor buffer set protocol, and stained with the indicated intracellular reagents. Analysis was performed to discriminate various T cell subpopulations.
Alexa Fluor Plus secondary antibodies—higher signal-to-noise ratio for your precious samples
When you perform cell imaging and fluorescence-based western blotting, you can’t afford to take risks with challenging samples for critical experiments. That’s why the Invitrogen Alexa Fluor dye–conjugated secondary antibodies you rely on for superior brightness and photostability are now available in an exclusive formula using our proprietary “Plus” dye chemistry. This unique dye chemistry coupled with high preadsorption enables Alexa Fluor Plus secondary antibodies to offer high signal-to-noise ratios and superior brightness.
See the visible difference:
The Invitrogen Alexa Fluor Plus 488 secondary antibody helps you see greater detail and shows a higher signal-to-noise ratio. Compare the conjugates: (A) Alexa Fluor 488 secondary antibody vs. (B) new Alexa Fluor Plus 488 secondary antibody.
Learn more about Alexa Fluor Plus secondary antibodies
Antibodies Learning Center
A new resource on thermofisher.com! The newly launched Antibodies Learning Center contains educational material designed to empower researchers and technicians with the background knowledge about antibody technologies necessary to develop, select, and/or use antibodies to advance their research.
This new resource center offers valuable information covering Cell Signaling Pathways, Antibody Methods, Application Notes and a related Resource Library.
BioProbes Journal Issue 74
BioProbes—Molecular Probes Journal of Cell Biology Applications
is a biannual publication that highlights a wide range of Thermo Scientific and Invitrogen cell biology products and applications. In issue 74 we feature articles on CRISPR, stem cell research, neuroscience, imaging, and flow cytometry.
eBioscience is now part of Thermo Fisher Scientific
In April 2016, eBioscience became part of Thermo Fisher Scientific with the acquisition of Affymetrix. Now, with the completion of our web integration, eBioscience reagents are available at thermofisher.com, providing a comprehensive and innovative portfolio of antibodies, fluorochromes, and reagents for multicolor flow cytometry, ideally suited for studies in immunology, oncology, cell biology, and stem cell biology.
In addition to multicolor flow cytometry reagents, the combined eBioscience and Invitrogen portfolio offers a broad spectrum of reagents for the analysis of cytokines, growth factors, and other soluble proteins. We offer bead-based multiplex immunoassays for flow cytometers, ready-to-use ELISA kits, matched antibody pairs, proteins, and standards, providing a complete solution for biological system analysis.
Visit thermofisher.com/ebioscience to explore the extensive cell analysis solutions.
For Research Use Only. Not for use in diagnostic procedures.