Bira biotin ligase

Manufactured by Avidity

Sourced in United States

The BirA biotin ligase is an enzyme that catalyzes the attachment of biotin to target proteins. This attachment, known as biotinylation, is a widely used technique in various biomedical and biochemical applications.

Automatically generated - may contain errors

Lab products found in correlation

Synergy neo2 plate reader, by Agilent Technologies (2 mentions) Nhs fluorescein, by Thermo Fisher Scientific (2 mentions) Dynabeads myone streptavidin t1, by Thermo Fisher Scientific (2 mentions) Superdex 200 column, by GE Healthcare (2 mentions) Pet22b, by GenScript (1 mentions) Superdex 200, by GE Healthcare (1 mentions) Thrombin, by Merck Group (1 mentions) Amicon ultra 0.5ml 100kda, by Merck Group (1 mentions) Superdex 200 size exclusion column, by GE Healthcare (1 mentions) Ni nta, by Qiagen (1 mentions) 293fectin reagent, by Thermo Fisher Scientific (1 mentions) Superdex 200 10 300 column, by GE Healthcare (1 mentions) Ovalbumin (ova), by Biosearch Technologies (1 mentions) Freestyle media, by Thermo Fisher Scientific (1 mentions) Ultra concentrators, by GE Healthcare (1 mentions) Ez link sulfo nhs lc lc biotin, by Thermo Fisher Scientific (1 mentions) Expi293 cell, by Thermo Fisher Scientific (1 mentions) Protein g sepharose 4 fast flow, by GE Healthcare (1 mentions) Fitc conjugated anti flag m2, by Merck Group (1 mentions) Liss rhod pe, by Avanti Polar Lipids (1 mentions)

13 protocols using bira biotin ligase

Isolation and Characterization of p53R175H-specific TCR

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

The isolation and characterization of p53R175H-specific TCR 6-11 from patients with colorectal cancer was described previously (14 (link)). Soluble TCR 6-11 for affinity measurement and structure determination was produced by in vitro folding from inclusion bodies expressed in E. coli, as described previously for other p53R175H-specific TCRs (19 (link)). Codon-optimized genes encoding the TCR α (residues 1–205) and β (residues 1–245) chains were synthesized and cloned into the expression vector pET22b (GenScript). An interchain disulfide (CαCys159–CβCys172) was engineered to increase the folding yield of TCR αβ heterodimer. Disulfide-linked TCR 6-11 was purified using sequential Superdex 200 and MonoQ FPLC columns (GE Healthcare).
Soluble HLA-A2 loaded with WT p53 peptide (HMTEVVRRC) or mutant p53R175H peptide (HMTEVVRHC) was prepared by in vitro folding of E. coli inclusion bodies as described (19 (link)). Correctly folded p53–HLA-A2 and p53R175H–HLA-A2 were purified using consecutive Superdex 200 and MonoQ columns. To produce biotinylated HLA-A2, a C-terminal tag (GGGLNDIFEAQKIEWHE) was attached to the HLA-A2∗0201 heavy chain. Biotinylation was carried out with BirA biotin ligase (Avidity).

Wu D., Gowathaman R., Pierce B.G, & Mariuzza R.A. (2022). T cell receptors employ diverse strategies to target a p53 cancer neoantigen. The Journal of Biological Chemistry, 298(3), 101684.

+ Open protocol

+ Expand

Fluorescent Csx30 Cleavage Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

To prevent labeling of Csx30-N amine side chains, we mutated eight lysine residues to arginine, and four lysines within the cleavage loop to alanine. Mutated and truncated Csx30 was purified as previously described except with HEPES buffer in all steps instead of Tris. Csx30 was biotinylated in vitro using the BirA biotin ligase (Avidity). Csx30 was incubated with NHS-Fluorescein (Thermo Fisher Scientific, #46409) on ice for 1 h before quenching with 200 mM Tris pH 7.5. Labeled Csx30 was purified using a Resource Q anion exchange column as before. Purified biotin-Csx30-FAM substrate was bound to MyOne Streptavidin T1 dynabeads (Thermo Fisher Scientific) in phosphate buffered saline (PBS) for 30 min at room temperature. The beads were washed 10 times with PBS supplemented with 0.1% bovine serum albumin and resuspended in PBS. In vitro reactions were performed as before and Dyneabeads were removed from the reaction using a magnetic stand. The supernatant, containing cleaved Csx30C, was transferred to 96-well plates and fluorescence measured using a Synergy Neo2 plate reader (BioTek) and subtracting the background signal from a well with no target RNA.

Strecker J., Demircioglu F.E., Li D., Faure G., Wilkinson M.E., Gootenberg J.S., Abudayyeh O.O., Nishimasu H., Macrae R.K, & Zhang F. (2022). RNA-activated protein cleavage with a CRISPR-associated endopeptidase. Science (New York, N.Y.), 378(6622), 874-881.

+ Open protocol

+ Expand

Biotinylation of Proteins with AviTag

Check if the same lab product or an alternative is used in the 5 most similar protocols

Proteins with AviTag were biotinylated as follows. Purified protein in 50 mm Bicine buffer (pH 8.3) at a final concentration of ∼1 mg/ml was incubated in the presence of 10 mm ATP, 10 mm magnesium acetate, 0.1 mm biotin, and BirA biotin ligase (Avidity) at 30 °C for 1 h and then placed at 4 °C overnight. The protein was then concentrated using an Amicon concentrator (molecular-weight cutoff, 10,000 Da), buffer-exchanged using PD-10 columns into storage buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, and 15% (v/v) glycerol), aliquoted, and flash frozen in liquid nitrogen.

Nimonkar A.V., Weldon S., Godbout K., Panza D., Hanrahan S., Cubbon R., Xu F., Trauger J.W., Gao J, & Voznesensky A. (2019). A lipoprotein lipase–GPI-anchored high-density lipoprotein–binding protein 1 fusion lowers triglycerides in mice: Implications for managing familial chylomicronemia syndrome. The Journal of Biological Chemistry, 295(10), 2900-2912.

+ Open protocol

+ Expand

Production and Characterization of HLA-DR Tetramers

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

HIS-tagged HLA-DRA/B1*0301, 0401, 0407, and 1501 protein monomers of wild type (wt) sequence or with L112W, S118H, V143M, T157I mutations (23 (link)) were produced by Hi5 insect cells and extracted from culture supernatant using Ni-NTA (Qiagen). HLA-DR monomers were biotinylated overnight at 4°C using BirA biotin ligase (Avidity) and purified by size exclusion chromatography using Superdex 200 size exclusion column (AKTA, GE Healthcare). Biotinylation was confirmed by gel-shift assay. Peptide exchange and tetramerization for wildtype and modified affinity-matured DR were performed using standard protocols as previously described (38 (link), 39 (link)). In brief, HLA-DR proteins were incubated with thrombin (Millipore) at room temperature for 3 – 4 hours and exchanged with peptides of interest in 50-fold excess at 37°C for 16 hours. Peptide-loaded HLA-DR monomers were incubated with fluorochrome-conjugated streptavidin at 4 – 5: 1 ratio for 2 min at room temperature, followed by a 15 min incubation with an equal volume of biotin-agarose slurry (Millipore). Tetramers were buffered exchanged into PBS, concentrated using Amicon ULTRA 0.5ml 100KDa (Millipore), and kept at 4 °C for no more than 2 weeks prior to use.

Pan Y.G., Bartolo L., Xu R., Patel B., Zarnitsyna V, & Su L. (2024). Differentiation marker-negative CD4+ T cells persist after yellow fever virus vaccination and contribute to durable memory. bioRxiv.

+ Open protocol

+ Expand

Preparation of Dual-Labeled SARS-CoV-2 Spike Trimers

Check if the same lab product or an alternative is used in the 5 most similar protocols

PreF (DS-Cav1) and postF (F ∆FP) trimers were produced with a single biotinylated C-terminal AviTag and then coupled to streptavidin-PE or streptavidin-APC⁵. Expression vectors containing each trimer with either a C-terminal 6x His-tag–AviTag or a C-terminal StrepTag II were co-transfected into FreeStyle 293-F cells at a 1:2 ratio. The protein was purified from the cell supernatant using Ni-nitrilotriacetic acid (NTA) resin to remove trimers lacking the 6× His-tag–AviTag and then purified over StrepTactin resin to remove those lacking the StrepTag II. Additonal wash steps removed trimers containing only one StrepTagII, and the remaining trimers containing two StrepTagII monomers and one 6× His-tag-AviTag monomer were then biotinylated using BirA biotin ligase (Avidity). The biotinylated proteins were separated from excess biotin by size-exclusion chromatography using a Superdex 200 column (GE Healthcare) in PBS. Dual-labeled tetramers were generated fresh for each experiment by incubating the trimers with premium-grade PE-labeled or APC-labeled streptavidin (Life Technologies) for 20 min on ice at a molar ratio of 4:1.

Shehata L., Wieland-Alter W.F., Maurer D.P., Chen E., Connor R.I., Wright P.F, & Walker L.M. (2019). Systematic comparison of respiratory syncytial virus-induced memory B cell responses in two anatomical compartments. Nature Communications, 10, 1126.

+ Open protocol

+ Expand

Purification and Labeling of Trimeric HA and Monomeric gp120

Check if the same lab product or an alternative is used in the 5 most similar protocols

Trimeric influenza hemagglutinin (HA) or and monomeric gp120 (resurfaced core 3) were purified as described (Weaver et al., 2016 (link); Wu et al., 2010 (link)). 293F cells grown in Freestyle media (Life Technologies) were transfected with 500 μg/L of HA or probe plasmid (293fectin™ Reagent, Life Technologies). At day 5, the culture was centrifuged (2,000 × g, 10 min) and the supernatant was filtered (VacuCap 8/0.2 μm filters, Pall Corporation) and loaded on Ni Sepharose FF resin (GE Healthcare) by gravity flow. The resin was washed (6 column volumes of BS containing 20mM imidazole) and then HA or gp120 was eluted in 500mM imidazole. The proteins were then concentrated (Amicon Ultra concentrators, 30kDa, cut off), and were separated by size exclusion FPLC using a Superdex 200 10/300 column (GE Healthcare). Trimeric HA or monomeric gp120 was then collected, concentrated and stored at −80°C. To construct a fluorescent version of HA, trimers bearing Avi tag (a site specific biotinylation sequence) were biotinylated using the commercial BirA biotin ligase (Avidity) and then labeled fluorescently with streptavidin conjugated APC (Life Technologies) as described previously (Weaver et al., 2016 (link)). Ovalbumin was purchased commercially (Biosearch Technologies).

Yousif A.S., Ronsard L., Shah P., Omatsu T., Sangesland M., Bracamonte Moreno T., Lam E.C., Vrbanac V.D., Balazs A.B., Reinecker H.C, & Lingwood D. (2020). The persistence of interleukin-6 is regulated by a blood buffer system derived from dendritic cells. Immunity, 54(2), 235-246.e5.

+ Open protocol

+ Expand

Fluorescent Csx30 Cleavage Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Duijm L.E., Guit G.L., Hendriks J.H., Zaat J.O, & Mali W.P. (1998). Value of breast imaging in women with painful breasts: observational follow up study. BMJ : British Medical Journal, 317(7171), 1492-1495.

+ Open protocol

+ Expand

ECoV-NC99 CTD+SD1 Protein Purification and Labeling

Check if the same lab product or an alternative is used in the 5 most similar protocols

The ECoV strain NC99 C-terminal domain and subdomain 1 (spike positions 316 to 687) were subcloned into pCMV/R with a synthetic signal peptide (MRPTWAWWLFLVLLLALWAPARG), and a C-terminal HRV3C cleavage site (LEVLFQ/GP) followed by an 8× HisTag and an AviTag (LNDIFEAQKIEWHE). The protein was expressed in Expi293 cells (ThermoFisher SCIENTIFIC, Waltham, MA, USA) and purified sequentially by Ni Sepharose excel beads (Cytiva, Marlborough, MA, USA) and gel filtration. The AviTag was then biotinylated using BirA biotin ligase (Avidity, Aurora, CO, USA) and cleaned of excess biotin by dialysis. Whole lyophilised venom from Dendroaspis polylepis was resuspended at 50 mg/mL, biotinylated at primary amines with EZ-Link™ Sulfo-NHS-LC-LC-Biotin (ThermoFisher SCIENTIFIC, Waltham, MA, USA) by manufacturer recommendations and purified from excess biotin. Biotinylated D. polylepis venom, polyclonal goat anti-horse (Pierce, Waltham, MA, USA), or ECoV-NC99 CTD+SD1 protein was labelled with AlexaFluor488 (AF488), phycoerythrin (PE), or allophycocyanin (APC) fluorescent streptavidin using saturating levels of biotinylated protein.

Wibmer C.K, & Mashilo P. (2022). Exploiting V-Gene Bias for Rapid, High-Throughput Monoclonal Antibody Isolation from Horses. Viruses, 14(10), 2172.

+ Open protocol

+ Expand

Purification and Functionalization of Ab62 mAb

Check if the same lab product or an alternative is used in the 5 most similar protocols

Ab62 mAb was purified from hybridoma supernatant using Protein G Sepharose 4 Fast flow (GE Healthcare Life Sciences, Pittsburgh, PA) per manufacturer protocol. FITC-conjugated anti-FLAG (M2) and M2-agarose were purchased from Sigma Aldrich (St. Louis, MO). Streptavidin (SA) was purchased from Calbiochem (Billerica, MA). Biotin ligase, Bir A, was obtained from Avidity (Aurora, CO). Lipids including DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), PC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, PG (1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1′-rac-glycerol)), Liss Rhod PE (L-α-Phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl), and malemide PEG₂₀₀₀-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] were purchased from Avanti Polar Lipids (Alabaster, AL). SATA (N-succinimidyl-S-acetylthioacetate) was from Pierce Biotechnology (Rockford, IL).

Greineder C.F., Hood E.D., Yao A., Khoshnejad M., Brenner J.S., Johnston I.H., Poncz M., Gottstein C, & Muzykantov V.R. (2016). Molecular Engineering of High Affinity Single-chain Antibody Fragment for Endothelial Targeting of Proteins and Nanocarriers in Rodents and Humans. Journal of controlled release : official journal of the Controlled Release Society, 226, 229-237.

+ Open protocol

+ Expand

Fluorescent Kinesin-1 Construct Purification

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Unlabeled and labeled (biotin and tetramethylrhodamine (TRITC)) lyophilized tubulin from porcine brain were purchased from Cytoskeleton. Neutravidin was purchased from Life Sciences (St. Louis, MO). Casein powder, glucose oxidase from Aspergillus niger, and bovine liver catalase were purchased from Sigma (St. Louis, MO). Mouse monoclonal anti-6xHis antibody was purchased from Abcam (Cambridge, MA). Biotinylated mouse anti-5xHis antibody was purchase from QIAGEN (Hilden, Germany). Mouse anti-tubulin β3 antibody from Bio-Rad Laboratories (Hercules, CA). We used a 6xHis-GFP-labeled truncated human kinesin-1 heavy chain construct (aa 1–560 (15 (link))) with an AviTag sequence susceptible to biotinylation added at the C-terminus (16 (link)). The protein was expressed in Escherichia coli, purified using cobalt resin from Clontech Laboratories (now Takara Bio USA, Mountain View, CA) (15 (link)). Biotin ligase BirA (Avidity) was used to biotinylate AviTag-kinesin (17 (link)).

Pyrpassopoulos S., Shuman H, & Ostap E.M. (2019). Modulation of Kinesin’s Load-Bearing Capacity by Force Geometry and the Microtubule Track. Biophysical Journal, 118(1), 243-253.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!