Ankyrons™ are next-generation target binding reagents provided by ProImmune that overcome many of the current constraints of research antibodies.
Figure: Domain comparison between Ankyrons and Antibodies. Ankyrons are highly stable recombinant single-domain binders. They can be directly labelled or detected via an epitope or biotin tag. Ankyrons can be engineered to combine them with further Ankyron or other domains much more easily than even recombinant Antibodies. Unlike Antibodies, Ankyrons do not have isotypes or any species origin.
Ankyrons disconnect from the animal-immune response paradigm of antibodies as binding reagents.
All Ankyrons are based on single domain ankyrin-repeat scaffold, selected in vitro by ribosome display for medium to high affinity from ProImmune’s synthetic Ankyron Teralibrary™ of around one trillion (~1012) clones. Therefore Ankyrons™ are by default recombinant sequence defined target binding molecules, unlike standard monoclonal antibodies. The exceptionally large starting clone number in our Teralibrary™ makes selecting Ankyrons more efficient than selecting antibody fragments from comparable antibody screening libraries in vitro.
Figure: Immunofluorescent analysis with Gabarap-specific Ankyrons (Clone AH50324). HeLa cells were infected with Plasmodium berghei for either 6 or 24 hours. The parasite was visualized with an antibody that recognises a protein in the parasitophorous vacuole membrane (PVM) in which the parasite resides in the host cell (UIS4 in magenta). Rows 1 and 2 are HeLa wild type cells. Rows 3 and 4 are staining of Gabarap knock-out HeLa cells. The Gabarap-specific Ankyron signal is in green. Gabaraps localize to the PVM previously determined from overexpression experiments using Gabarap-GFP. Data was kindly provided by Dr. Jacqueline Schmuckli and Prof. Volker Heussler, Institute of Cell Biology, University of Bern, Switzerland
Cells contain various membrane-bound organelles, each with specific proteins related to their function. We have made available a panel of Ankyrons that detect these organelle-specific proteins, allowing you to identify the subcellular location of your protein of interest and investigate the role it may play within the cell. In the images below, we focus on HeLa cells, imaging with epifluorescence microscopy at 60 X magnification and detecting bound Ankyrons with an APC-conjugated secondary antibody that recognizes the V5 tag on the Ankyrons (red), counterstaining the nuclei with DAPI (blue).
Ankyrons that recognize the chaperone proteins calreticulin and calnexin provide excellent markers for the endoplasmic reticulum. The endoplasmic reticulum is involved in synthesis of proteins, lipids and steroids and is continuous with the outer membrane of the nuclear envelope, hence, is apparent in close proximity to the nucleus.
Figure: Endoplasmic reticulum: Calreticulin (ANK00703_2; Clone AHJ30446)
Figure: Endoplasmic reticulum: Calnexin (ANK00865_4; Clone AY40204)
The Ankyron binding RCAS1 (Receptor binding Cancer Antigen expressed on SiSo cells, also known as Estrogen Receptor-Binding Fragment-Associated Gene 9, EBAG9) can be applied as a Golgi complex marker. RCAS1 is a protein that regulates vesicle formation, secretion and protein glycosylation. The Golgi complex receives proteins from the ER, then processes and sorts them.
Figure: Golgi complex: RCAS1 (ANK01013_4; Clone BA40122)
By using an Ankyron that binds the tetraspanin CD63, late endosomes, lysosomes and secretory vesicles within the cell and in perinuclear compartments can be labelled.
Figure: Lysosomes (and perinuclear) staining: CD63 (ANK01427_1; Clone AW40513)
CD44 is an almost ubiquitously expressed protein on cells of epithelial, mesenchymal and haematopoietic origin. Here, the CD44-specific Ankyron is used as an effective marker for the plasma membrane. CD44 is also a useful uropod marker in migratory cells.
Figure: CD44 (ANK00521_1; Clone BV40957)
Ankyron staining against Histone H3.1 specifically stains nuclei, and thus is co-localizing with DAPI.
Figure: Nucleosomes (ANK00169_5; Clone AE40386)
We also provide Ankyrons targeting LAMP-1 (an alternative lysosomal marker), GOPC (an alternative Golgi marker), HSP60 (a mitochondrial marker) and the exosome marker CD9, and will be expanding our range in the near future.
Figure: Flow cytometry staining with HIF-1 alpha transcription factor-specific Ankyron (Clone AW40110) on NK cells from C57BL/6 mice. C57BL/6 mice were injected with MC38 tumour cells. NK cells in tumors and spleen were isolated and stained for HIF-1α. Staining with Ankyron clone AW40110 clearly shows low HIF-1α expression in the spleen which is elevated in NK cells from tumors. Data was kindly provided by Prof. Dave Withers, University of Birmingham, United Kingdom
Watch a brief summary of the Ankyron technology
*Contact us to discuss your options, including our reduced cost or even free of charge priority access program or Request a Quote (and we will respond within 1 working day).
You will be able to get Ankyrons for almost any target with and label, custom-configured every time with a plethora of formatting and customization options, including for bi-and multi-specific molecules, combined with a full range of labeling options, covering most types of available fluorochromes and other labels.
Any Ankyron is always available with any tag and label option we offer as standard.
Unlike catalog research antibodies, our catalog Ankyrons can be customized rapidly to your evolving requirements. Ankyrons help you move away from the primary/secondary antibody paradigm that still exists for most antibodies except those for the most common targets.
Our catalog range includes more than 20,000 clones for over 1,300 target proteins so far.
Our catalog single-domain Ankyrons™ come with a 6His-tag and a V5 epitope tag by default. The customer has the choice of swapping the V5 epitope tag to the following alternative tags:
Alternative tags: Flag, C-Myc, HA, StII(WSHPQFEK), Biotin tag.
In order to label Ankrons™ We have developed Alphalux™ labeling technology for consistent labeling of all Ankyrons with the following:
Standard covalent labels: Alphalux™ 405, 488, 532, 555, 647, 680, R-PE, APC, HRP
Note: Alphalux™ 405, 488, 532, 555, 647, 680 are spectrally equivalent to Alexafluor® colors.
Standard streptavidin (SA) labels via 1:1 conjugation of biotinylated Ankyron to:
SA-PE, SA-APC, SA-PE-Cy5, SA-PE-Cy7, SA-APC-Cy7, SA-HRP
Ankyrons can be conveniently labeled to order with any standard covalent fluorescent label or with any other standard streptavidin or avidin conjugate for minimal extra costs. It won’t take much longer either. If the label you want is not in the list above, please contact us for rapid turnaround no-obligation custom quote. Have it your way, every time!
With Ankyrons the primary vs secondary and catalog vs custom concepts either merge or become redundant.
Ankyrons blur the divide that has existed between traditional catalog and custom antibody products. Even if you don’t have the target protein you are interested in we may be able to make it, or a recombinant fragment thereof, for you either at no or low cost, if it is a publicly known target of general interest. We offer a privileged access program to free of charge custom made reagents, including raising Ankyrons to new targets in areas that are of sufficient interest to ProImmune. If you are interested in discussing this program, please contact us.
Find out more about custom Ankyrons.
Ankyron Guided Selection is like a superpower allowing you to drill into to the specificity you need
Ankyrons are selected directly in vitro from our Teralibrary™. In our discovery process we can easily screen Ankyron binders for selective reactivity with one type of target over closely related versions of the same target. Prominent examples include peptide-specific Ankyrons to peptide-MHC molecules and idiotype specific Ankyrons to antibodies, such as drug antibodies.
Figure: Guided selection discriminates which Ankyrons bind to specific variants of a molecule.
With Ankyrons (almost everything) comes within reach!
Research antibodies exist for many human targets and to a much lesser extent for targets from other species. Many gaps in coverage however still exist for many important research areas, due to the cost of raising antibodies, especially monoclonal and recombinant antibodies. Ankyrons are fundamentally suited for efficient and cost effective high throughput discovery, including to challenging targets such as peptide-MHC molecules. Contact us for the best options for custom discovery.
Humans exist among a universe of species. We need to find out more about them to find out more about ourselves. Affordable high-quality affordable new catalog and custom Ankyrons help you to explore more, faster, and with more confidence.
New Ankyron discovery is at the heart of the Ankyron story. Supporting discovery in key interest areas requires access to high quality antigen that is specifically configured for Ankyron discovery. To support this effort we have developed an internal high-throughput antigen provisioning platform which we call AnkyronX. AnkyronX can deliver hundreds of proteins for target discovery quickly and cost-effectively.
Ankyrons are automatically selected for high affinity
When we discover new target binding Ankyrons from our library we automatically only select for high affinity binders through our process. The typical affinity of most Ankyrons for their target is therefore at least in the nanomolar range and often we achieve picomolar affinity.
Figure: Affinities for Ankyrons can be determined for example by solution equilibrium titration assay. Ankyrons A1, A2, A3, selected from the Teralibrary for binding to the immuno-oncology target PDL1 were determined to have the following affinities: A1: 0.7 pM, A2: 160 pM, A3: 600 pM.