Choosing Between an Amine-Labeled or an Avi-tag Biotinylated Protein
Biotinylated proteins can be powerful tools for assessing protein-protein interactions and screening antibody or small molecule libraries for potential therapeutics. Avi-tag recombinant proteins contain an extra 15 amino acid sequence, the Avi-tag, typically fused to the N- or the C-terminus of protein. This Avi-tag peptide is recognized by BirA ligase which enzymatically attaches a biotin molecule to a single lysine residue within the Avi-tag sequence. As a result, Avi-tag proteins are biotinylated in a site-specific manner, allowing for consistent, uniform protein biotinylation. This provides enhanced control of protein orientation in binding assays, and since the Avi-tag is located on the end of the protein, the effect on the activity of the protein is typically minimal. The result is a biotinylated protein that maintains the same characteristics as the unlabeled protein.
In contrast to Avi-tag biotinylated proteins, amine-labeled biotinylated proteins are generatated by chemical biotinylation. This labels the protein on amine groups found at the N-terminus and in lysine residues throughout the protein, resulting in multiple biotins being incorporated per protein. The result is a biotinylated protein with a high binding efficiency to avidin or streptavidin and high signal strength.
As biotin is a relatively small molecule, it typically does not affect the activity of the protein, but our in-house scientists rigorously test both our amine-labeled and Avi-tag biotinylated proteins to ensure that they exhibit the same level of bioactivity as the unlabeled protein. Additionally, the lot-to-lot consistency of all our proteins are routinely evaluated. Each new lot is tested side-by-side with previous lots and with a master lot, so you don’t have to worry whether your results will be reproducible over time.
Explore our large selection of R&D Systems™ amine-labeled and Avi-tag biotinylated proteins to find the right protein for your assay.
R&D Systems Biotinylated Proteins Video
This video describes how R&D Systems amine-labeled and Avi-tag biotinylated recombinant proteins are produced and the advantages associated with either chemical or enzymatic biotinylation.
Advantages of Avi-Tag Biotinylated Proteins
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Consistent, highly specific labeling. A single biotin molecule is enzymatically added to a lysine residue in the Avi-tag by BirA biotin ligase, resulting in the generation of a homogeneous product.
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Uniform orientation of the protein. When bound to a streptavidin-coated surface, the orientation of the Avi-tag biotinylated protein will be uniform due to the precise control over biotinylation.
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Similar bioactivity as the unlabeled protein. All R&D Systems biotinylated proteins are rigorously tested to ensure that they exhibit the same level of bioactivity as the unlabeled protein.
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Lot-to-lot consistency. Each new lot is tested side-by-side with previous lots and with a master lot, so you don’t have to worry whether your results will be reproducible over time.
Avi-tag Proteins – Products by Molecule
Advantages of Amine-Labeled Biotinylated Proteins
• High signal strength. Chemical biotinylation labels the protein on amine groups found in lysine residues throughout the protein and at the N-terminus, resulting in multiple biotins being incorporated per protein.
• Similar bioactivity as the unlabeled protein. All R&D Systems biotinylated proteins are rigorously tested to ensure that they exhibit the same level of bioactivity as the unlabeled protein.
• Lot-to-lot consistency. Each new lot is tested side-by-side with previous lots and with a master lot, so you don’t have to worry whether your results will be reproducible over time.
Amine-Labeled Biotinylated Proteins - Products by Molecule
R&D Systems Biotinylated Proteins Exhibit the Same Levels of Bioactivity as the Equivalent Unlabeled Proteins
Unlabeled and Avi-tag Biotinylated Recombinant Human PD-L1/B7-H1 Display Comparable Bioactivity. Human T lymphocytes were treated with the indicated concentrations of either unlabeled Recombinant Human PD-L1/B7-1 Fc Chimera (R&D Systems, Catalog # 156-B7; green line) or Avi-tag Biotinylated Recombinant Human PD-L1/B7-H1 Fc Chimera (R&D Systems, Catalog # AVI156; orange line). IL-2 secretion was measured in cell culture supernatants using the Human IL-2 Quantikine® ELISA Kit (R&D Systems, Catalog # D2050). The similarity in the activities of the two proteins highlights that the Avi-tag biotinylated protein is fully functional.
Unlabeled and Biotinylated Recombinant Human TNF-alpha Display Comparable Bioactivity in an Assay Measuring Cytotoxicity in L-929 Mouse Fibroblasts. L-929 mouse fibroblast cells were treated with the indicated concentrations of either unlabeled Recombinant Human TNF-alpha (R&D Systems, Catalog # 210-TA; green line) or Biotinylated Recombinant Human TNF-alpha (R&D Systems, Catalog # BT210; orange line), in the presence of the metabolic inhibitor, actinomycin D, and cytotoxicity was assessed. The ED50 for this effect is 25-100 pg/mL. The similarity in the activities of the two proteins highlights that the biotinylated protein is fully functional.
Unlabeled and Biotinylated Recombinant Human PDGF-BB Display Comparable Bioactivity in an Assay Measuring Proliferation of NR6R-3T3 Mouse Fibroblast Cells. NR6R-3T3 mouse fibroblast cells were treated with the indicated concentrations of either unlabeled Recombinant Human PDGF-BB (R&D Systems, Catalog # 220-BB; green line) or Biotinylated Recombinant Human PDGF-BB (R&D Systems, Catalog # BT220; orange line) and cell proliferation was assessed. The similarity in the activities of the two proteins highlights that the biotinylated protein is fully functional.
R&D Systems Biotinylated Proteins Are Rigorously Tested to Ensure Lot-to-lot Consistency
R&D Systems Avi-tag Biotinylated Recombinant Human PD-L1/B7-H1 Displays High Lot-to-Lot Consistency. Three independent lots of Avi-tag Biotinylated Recombinant Human PD-L1/B7-H1 (R&D Systems, Catalog # AVI156) were tested for their ability to bind to Recombinant Human PD-1 (R&D Systems, Catalog # 1086-PD), which was coated at 1 ug/mL. Avi-tag Biotinylated Recombinant Human PD-L1/B7-H1 bound with an ED50 of 8-48 ng/mL. Each trace shown on the graph represents data obtained from Avi-tag Biotinylated Recombinant Human PD-L1/B7-H1 from a different manufacturing run to show the lot-to-lot consistency of the proteins.
Analysis of the Binding Properties of R&D Systems Avi-tag Biotinylated Proteins
Affinity Measurements and Binding Kinetics of the PD-1:PD-L1 Interaction by Surface Plasmon Resonance. Sensorgram data of captured Avi-tag Biotinylated Recombinant Human PD-L1 His tag (R&D Systems, Catalog # AVI9049) binding to Recombinant Human PD-1 His tag (R&D Systems, Catalog # 8986-PD). The corresponding overlaid kinetic fits with the residual plot shown below. The concentration of Recombinant Human PD-1 His-tag ranged from 3.2 nM to 13.2 μM. The corresponding steady state affinity fit is shown below. The experiment was performed on a BiacoreT200, GE Healthcare.
Affinity Measurements and Binding Kinetics of the CTLA-4:B7-1 Interaction by Surface Plasmon Resonance. Sensorgram data of captured Avi-tag Biotinylated Recombinant Human CTLA-4 Fc Chimera (R&D Systems, Catalog # AVI7268) binding to Recombinant Human B7-1 Fc Chimera (R&D Systems, Catalog # 10133-B1). Briefly, Avi-tag Biotinylated Recombinant Human CTLA-4 Fc Chimera was captured at a low coupling density to the active flow cell via the Avi-tag biotin. Recombinant Human B7-1 Fc Chimera at a concentration range between 82 pM and 21 nM was flowed over both active and uncoupled reference flow cells at each concentration. Kinetic sensorgrams were fit to a 1:1 binding model to determine the binding kinetics and affinity, with an interaction affinity of KD=0.2511 nM. The corresponding overlaid kinetic fits with the residual plot shown below. The corresponding steady state affinity fit is shown at the bottom. The experiment was performed on a Biacore T200, GE Healthcare.
Affinity Measurements and Binding Kinetics of the CD47:SIRP alpha Interaction by Surface Plasmon Resonance. Sensorgram data of captured Avi-tag Biotinylated Recombinant Human CD47 Fc tag (R&D Systems, Catalog # AVI4670)binding to Recombinant Human SIRP alpha His tag (R&D Systems, Catalog # 9378-SA). The corresponding overlaid kinetic fits with the residual plot shown below. The concentration of Recombinant Human SIRP alpha His-tag ranged from 6.85 nM to 3.51 μM. The corresponding steady state affinity fit is shown at the bottom. The experiment was performed on a Biacore T200, GE Healthcare.
Affinity Measurements and Binding Kinetics of the CD155/PVR:TIGIT Protein Interaction by Surface Plasmon Resonance. Sensorgram data of captured Avi-tag Biotinylated Recombinant Human CD155/PVR Fc Chimera (R&D Systems, Catalog # AVI9174) binding to Recombinant Human TIGIT His-tag (R&D Systems, Catalog # 9525-TG). The corresponding overlaid kinetic fits with the residual plot shown below. The concentration of Recombinant Human TIGIT His-tag ranged from 0.2 nM to 400 nM. The corresponding steady state affinity fit is shown at the bottom. The experiment was performed on a Biacore T200, GE Healthcare.
Additional Resources
Wall Poster: Immune Checkpoint Targets
Wall Poster: Immune Checkpoint Targets
Immune checkpoint molecules deliver signals that control the activities of a variety of immune cell types and are some of the most highly investigated targets for immuno-oncology research. Use our new wall poster to explore the current and emerging immune checkpoint molecules being investigated as potential cancer immunotherapy targets.
eBook: Current and Emerging Immune Checkpoint Targets for I-O Research
eBook: Current and Emerging Immune Checkpoint Targets for I-O Research
Immune checkpoint proteins play a central role in regulating the activities of different immune cell types and represent some of the most promising targets for cancer immunotherapy. This eBook highlights some of the immune checkpoint molecules that are being investigated as immuno-oncology targets, and the products that we offer for studying these molecules.
Wall Poster: A Look Inside a Tumor
Wall Poster: A Look Inside a Tumor
The tumor microenvironment (TME) plays a central role in inhibiting anti-tumor immune responses. Use our poster to learn about the key mechanisms by which tumor cells, tumor-derived exosomes, regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) mediate immunosuppression in the tumor microenvironment.
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Custom Protein Services
If you can’t find the protein that you need on our website, contact us. From scratch protein development to customizing a protein from our catalog, our custom services team will create the protein that fits your experimental needs.