Ankyrons™ are next-generation target binding reagents that overcome many of the current constraints of research antibodies. They are small, highly stable, recombinant single-domain proteins based on an ankyrin repeat scaffold.
All Ankyrons are selected in vitro by ribosome display for medium to high affinity from our Teralibrary™ of around one trillion clones. Ankyrons™ are by default recombinant sequence defined target binding molecules. Our Teralibrary™ makes selecting Ankyrons more efficient than selecting antibody fragments from comparable antibody screening libraries in vitro.
Figure. Cadherin-1 (E-Cadherin) Ankyron clone ANK00519_1 staining on HeLa cells and HCT116 cells. The cell-type specific presence of E-Cadherin on the surface HCT116 cells is clearly visible (red) with no background staining on E-Cadherin-negative HeLa cells. V5-tag labelled Ankyron was detected with APC labelled Anti-V5-Tag. Nuclei stained with DAPI (blue).
Ankyrons provide excellent quality staining for immunofluorescence. Their small size means the fluorophore is in close proximity to the binding site enabling high resolution imaging. Furthermore, their small size allows for less aggressive permeabilization of cells and shorter staining and washing steps. Compared to immunofluorescence staining protocols with antibodies, the standard Ankyron protocol is significantly faster saving you over an hour in lab time. View our Ankyron protocols here.
Figure. VCAM-1 (Vascular Cell Adhesion Molecule 1) is absent from resting blood endothelia but upregulated upon inflammation. VCAM-1 engages with its ligand VLA4 (integrin α4β1) on leukocytes, to mediate adhesion and capture from blood flow, prior to extravasation and migration into inflamed tissues. Human umbilical vein endothelial cells (HUVEC) in culture do not constitutively express VCAM-1. VCAM-1 expression can be induced by stimulation with the pro-inflammatory cytokine TNFα. Here, we stain HUVEC using one of our five Ankyron™ clones (ANK00527_1) against VCAM-1 (red), counterstaining nuclei with DAPI (blue).
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: Nucleosomes (ANK00169_5; Clone AE40386) Ankyron staining against Histone H3.1 specifically stains nuclei, and thus is co-localizing with DAPI.
Figure: Immunofluorescent analysis with Gabarap-specific Ankyrons (Clone AH50324). HeLa cells were infected with Plasmodium berghei and 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). Row 1 is HeLa wild type cells. Row 2 is 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
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
As an example, APRIL-specific Ankyron™ can be used effectively in immunohistochemistry of formalin-fixed paraffin-embedded tissue, as shown below in human colon, where APRIL is believed to have a role in intestinal epithelial – immune cell crosstalk.
Figure: Immunohistochemical analysis of paraffin-embedded human colon, using 10 μg/ml V5-tagged Ankyron TNF13_HUMAN_ANK03668_3 and anti-V5 HRP (1:500), detecting with DAB (brown) and counterstaining nuclei with haematoxylin (blue-purple). Heat-mediated antigen retrieval with Leica Citrate buffer (pH6).
Background APRIL (a proliferation-inducing ligand) is a member of the tumour necrosis factor (TNF) ligand superfamily (TNFSF13, also known as CD256) and plays a key role in humoral immunity, through regulating B-cell maturation, proliferation and survival by interacting with receptors TACI (TNFRSF13B) and BCMA (TNFRSF17). Under pathological conditions, APRIL is involved in the development of systemic autoimmune diseases, such as lupus erythematosus and rheumatoid arthritis. Unlike many other ligands of the TNF superfamily, APRIL can also stimulate growth of cancer cells and may be over-expressed during neoplasia. Thus, it is a useful diagnostic marker and an attractive therapeutic target in autoimmune diseases, immunoglobulin disorders and cancer.
*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 7,500 clones for over 1,700 target proteins to date.
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.
Figure: Clathrin is the major component of the polyhedral lattice on the cytoplasmic face of membranes encompassing coated pits and vesicles, with well-characterized roles in endocytosis. It also contributes to stabilizing the kinetochore fibres of the mitotic spindle, acting as an inter-microtubule bridge. Here, we image the spindle of a mitotic human umbilical vein endothelial cell (HUVEC) using a GFP-tagged Ankyron™ specific to Clathrin Heavy Chain 1 (ANK03865_1, green), counterstaining nuclei with DAPI (blue) and capturing images by widefield microscope.
Figure: Here we have followed the translocation of NFkB p65 subunit from cytoplasm in resting, unstimulated human umbilical vein endothelial cells (HUVECs, upper panels) to the nucleus following stimulation with TNFalpha (lower panels) using Ankyron clone AW40914 targeting TF65, counterstaining with phalloidin to identify actin filament (green), DAPI (blue) to identify nuclei and capturing images by widefield microscope.
