DRG neurons expressing AviTag-NF186 at DIV 5–8 were rinsed with biotinylation media (1% FBS C medium, 5 mM Mg2+, and 10 mM Hepes) then incubated with the biotinylation solution containing 0.3 µM BirA ligase, 10 µM d-biotin, and 1 mM ATP (Avidity) in biotinylation medium for 10 min at 37°C. Cultures were rinsed with 1% FBS C medium with 10 mM Hepes and incubated with 5 µg/ml streptavidin–Alexa Fluor-568 (Invitrogen) or 4 µg/ml pHrodo Red Avidin (Invitrogen) for 5 min at RT. After washing, cultures were live imaged. In other studies, DRG neurons were incubated with 50 nM LysoTracker Red DND-99 (ThermoFisher Scientific) in NB medium for 1 h at 37°C.
D biotin
Manufactured by Avidity
Sourced in United States
D-biotin is a water-soluble vitamin that serves as a cofactor for several enzymes involved in carboxylation reactions. It plays a crucial role in energy metabolism, cell growth, and the synthesis of fatty acids.
Automatically generated - may contain errors
Lab products found in correlation
Pe streptavidin, by Merck Group (2 mentions)
Facsaria fusion, by BD (2 mentions)
Dcode dextramer pe reagents, by Immudex (2 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Phrodo red avidin, by Thermo Fisher Scientific (1 mentions)
Streptavidin alexafluor 568, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by Avidity (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Pbiracm, by Avidity (1 mentions)
Potassium chloride (kcl), by Thermo Fisher Scientific (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by GoldBio (1 mentions)
Norland 65, by Norland Products (1 mentions)
Amyl acetate, by Electron Microscopy Sciences (1 mentions)
Hepes, by GoldBio (1 mentions)
Mini extruder, by Cytiva (1 mentions)
Plusone repel silane es, by GE Healthcare (1 mentions)
Whatman, by Cytiva (1 mentions)
Hitrap, by Cytiva (1 mentions)
12 protocols using d biotin
1
Biotinylation and Labeling of DRG Neurons
2
Preparation and Characterization of HBV-Peptide Tetramers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
WT H-2Kd and the three H-2Kd mutant-restricted tetramers of HBV-peptide were prepared as previously described (Liu et al., 2012a (link); Zhang et al., 2018 ; Zhou et al., 2006 (link)). Briefly, recombinant H-2Kd/peptide complexes were purified and then biotinylated by incubation with D-biotin, ATP, and the biotin protein ligase BirA (Avidity) at 4 °C for 12 h. The biotinylated H-2Kd was further purified by gel filtration to remove free biotin, and then the multimers were produced by using PE-streptavidin (Sigma). Cells from the subjects were stained with PE-tetramer and FITC-conjugated anti-CD8 antibody. All samples were analyzed with a FACSCalibur flow cytometer (BD Biosciences) after staining.
3
Dextramer-based single-cell RNA sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were thawed in RPMI media and centrifuged at 350×g for 10min at 4°C. Cells were washed with Washing Buffer (PBS, pH 7.4 + 5% FBS), centrifuged, and the cell pellet was resuspended in Stain Buffer (PBS, pH 7.4 + 5% FBS + 0.1g/L Herring sperm DNA). Then, we added 0.2 μl 100μM d-Biotin (Avidity LLC, Aurora, CO) per included dextramer and added 2μl of desired dCODE™ Dextramer®-PE reagents (Immudex, Denmark), including the negative control for allele and random dextramers (Table 1 , dextramers #1–4 for cHCV and rHCV samples, and dextramer #1, #5–6 for DAA therapy samples). After 10 min of incubation in the dark, we added the antibody cocktail for CD8+ T cell staining (CD4-APC, CD3-FITC, CD8-V500, and CD56-V450) and incubated for 30 min at 4°C in the dark. We sorted the CD3+ CD8+ Dextramer+ cells using BD FACSAria™ Fusion into 90% FBS solution at 4°C (Figure 1A , Supplementary Figure 1 ). Sorted cells were centrifuged and resuspended in 50μl of PBS with 0.04% BSA for scRNAseq partitioning.
4
Dextramer-based single-cell RNA sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were thawed in RPMI media and centrifuged at 350×g for 10min at 4°C. Cells were washed with Washing Buffer (PBS, pH 7.4 + 5% FBS), centrifuged, and the cell pellet was resuspended in Stain Buffer (PBS, pH 7.4 + 5% FBS + 0.1g/L Herring sperm DNA). Then, we added 0.2 μl 100μM d-Biotin (Avidity LLC, Aurora, CO) per included dextramer and added 2μl of desired dCODE™ Dextramer®-PE reagents (Immudex, Denmark), including the negative control for allele and random dextramers (Table 1 , dextramers #1–4 for cHCV and rHCV samples, and dextramer #1, #5–6 for DAA therapy samples). After 10 min of incubation in the dark, we added the antibody cocktail for CD8+ T cell staining (CD4-APC, CD3-FITC, CD8-V500, and CD56-V450) and incubated for 30 min at 4°C in the dark. We sorted the CD3+ CD8+ Dextramer+ cells using BD FACSAria™ Fusion into 90% FBS solution at 4°C (Figure 1A , Supplementary Figure 1 ). Sorted cells were centrifuged and resuspended in 50μl of PBS with 0.04% BSA for scRNAseq partitioning.
5
Recombinant Mouse IL-38 Biotinylation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant mouse IL-38 was expressed as an N-terminal fusion protein with hexa-histidine followed by ubiquitin (His-Ub) and concluded at the C terminus with an Avi-tag (GLNDIFEAQKIEWHE) for site-specific biotinylation. The protein was expressed in BL21 (DE3) co-transformed with pBirAcm (Avidity, L.L.C., USA) plasmid that encodes for biotin-ligase A. To ensure sufficient biotinylation, D(+)-biotin (Roth) was added to the expression medium to a final concentration of 100 mM. Expression and purification were done as before, but without cleavage of the His-Ub-tag by USP2 (Mora et al., 2016) . The resulting protein fractions after elution (800 mM NaCl, 400 mM Imidazole, 50 mM Na3PO4, 1 mM b-mercaptoethanol and 4% glycerol) were dialyzed in PBS (137 nM NaCl, 2.7 nM KCl, 10 nM Na2HPO4 and 1.8 nM KH2PO4 at pH 7.4).
6
Reconstitution of Microtubule Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GTP and paclitaxel (Taxol) were purchased from Cytoskeleton (Denver, CO). Guanosine-5′-[(α,β)-methyleno]triphosphate, sodium salt (GMPCPP), the nonhydrolyzable analog of GTP, was purchased from Jena Bioscience (Jena, Germany). Piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), ATP, and glucose were purchased from Sigma (St. Louis, MO). HEPES and dithiothreitol (DTT) were purchased from GoldBio (St Louis, MO). EGTA, MgCl2, and KCl were purchased from Thermo Fisher Scientific (Waltham, MA). Sterile collodion (nitrocellulose) 2% in amyl acetate and amyl acetate were purchased from Electron Microscopy Sciences (Hatfield, PA). d-Biotin was purchased from Avidity (Aurora, CO). Chloroform solutions of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl), mini extruder, Nuclepore track-etch membrane (50 nm) from Whatman, and filter support disks 10 mm from Whatman were purchased from Avanti Polar Lipids (Alabaster, AL). High vacuum grease, silicone elastomer base (184 Sylgard), and the corresponding elastomer curing agent were manufactured by Dow Corning (Midland, MI). 2% Dimethyldichlorosilane in octamethylcyclooctasilane (PlusOne Repel-Silane ES) was purchased by GE Healthcare (Chicago, IL). Optical adhesive Norland 65 was purchased from Norland Products (Cranbury, NJ).
7
Biotinylation and Tetramerization of HLA-B*18:01
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HLA‐B*18:01 heavy chain with a BirA enzyme tag was obtained as described above (GenScript). Subsequently, after anion exchange, the protein complex solution was desalted using a HiTrap column (Cytiva). The monomeric protein was combined with 10% v/v 0.5 m bicine pH 8.3 (Avidity, Aurora, USA), 10% v/v 100 mM ATP, 100 mM magnesium acetate, 500 μm D‐biotin (Avidity) and 5 μg of biotin protein ligase per mg of protein. After overnight incubation at 4 °C, the solution was loaded onto a size exclusion (Superdex S200 10/300, Marlborough, USA) column. The pure protein fraction was pooled and the amount of biotinylated protein monomer was measured via native gel electrophoresis. Each peptide‐HLA‐B*18:01 monomer was conjugated at an 8:1 peptide‐HLA to Streptavidin‐PE molar ratio (BD Biosciences, Franklin Lakes, USA) to form tetramers of HLA‐B*18:01 presenting M15, NS2111, PB1177 or NP219 (hereafter, tetramers and tetramer+ cells are referred to as B18/M15, B18/NS2111, B18/PB1177 or B18/NP219, respectively).
8
UV-Mediated Peptide Exchange for MHC Multimers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Soluble biotinylated Class-I MHC monomers containing a UV-cleavable peptide in their binding groove were produced in-house according to published protocols77 (link)
,78 (link) and stored at −80°C. The UV-dependent peptide exchange (25 μg/mL UV-monomer and 50 μg/mL peptide diluted in PBS) was performed at a wavelength of 366 nm for 1 h at 4°C. 24 h after UV-exchange, the following streptavidin-tagged fluorochromes were added at optimized ratios to the pMonomer solution: SA-PE (Invitrogen), SA-APC (Invitrogen), SA-BV421 (Biolegend), SA-BV605 (Biolegend), SA-PE-Cy7 (Biolegend), SA-PE-CF594 (BD Biosciences), SA-PE-Cy5 (BD Biosciences), SA-APC-R700 (BD Biosciences), SA-BB790-P (BD Biosciences; prototype kindly provided by Bob Balderas). To block unoccupied binding sites, D-biotin (20 μM; Avidity) was added 24 h after multimerization. Plates were stored in the dark at 4°C until use.
,78 (link) and stored at −80°C. The UV-dependent peptide exchange (25 μg/mL UV-monomer and 50 μg/mL peptide diluted in PBS) was performed at a wavelength of 366 nm for 1 h at 4°C. 24 h after UV-exchange, the following streptavidin-tagged fluorochromes were added at optimized ratios to the pMonomer solution: SA-PE (Invitrogen), SA-APC (Invitrogen), SA-BV421 (Biolegend), SA-BV605 (Biolegend), SA-PE-Cy7 (Biolegend), SA-PE-CF594 (BD Biosciences), SA-PE-Cy5 (BD Biosciences), SA-APC-R700 (BD Biosciences), SA-BB790-P (BD Biosciences; prototype kindly provided by Bob Balderas). To block unoccupied binding sites, D-biotin (20 μM; Avidity) was added 24 h after multimerization. Plates were stored in the dark at 4°C until use.
9
SARS-CoV-2 Spike Protein Biotinylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full‐length SARS‐CoV‐2 Spike (2P‐stabilized, C‐terminal Histidine/Avi‐tagged) was obtained from BEI resources (Manassas, VA, Cat. NR53524) and biotinylated using a BirA ubiquitin ligase (Avidity, Aurora, CO, Cat. Bir500A) following the manufacturer's recommended protocol. Biotinylated spike protein was purified using a 40 K molecular weight cut‐off (MWCO) 2 ml Pierce Zeba™ desalting column (Thermo Fisher Scientific, Waltham, MA, Cat. 87768), and mixed with streptavidin BV421 (BD, Cat. 563259) or streptavidin allophycocyanin (APC) (BD Cat. 554067) separately at a 20:1 ratio (~6:1 molar ratio) as previously described [3 (link)]. Tetramerized Spike probes were stored at 4°C until use. Just prior to probe staining, spike protein probes were added one‐by‐one to FACS wash buffer (1x PBS, 2% fetal bovine serum) containing 5 μM free d‐biotin (Avidity, Cat. Bir500A). Both streptavidin‐fluor conjugates were used to stain dimethyl sulfoxide (DMSO) control samples to further verify the absence of significant frequencies of nonspecific streptavidin‐binding B cells.
10
Purification and Biotinylation of Sal-1 DBP-II
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BirA‐tagged Sal‐1 DBP‐II was prepared as described previously.5 , 6 , 7 , 19 Briefly, inclusion bodies were solubilized in 6 M guanidinium hydrochloride and refolded via rapid dilution into 400 mM l ‐arginine, 50 mM Tris (pH 8.0), 10 mM EDTA, 0.1 mM PMSF, 2 mM reduced glutathione, and 0.2 mM oxidized glutathione. Refolded protein was captured on SP Sepharose Fast Flow resin (GE Healthcare) and further purified by SEC (GF200; GE Healthcare) into 10 mM Hepes (pH 7.4) and 100 mM NaCl.
BirA‐tagged Sal‐1 DBP‐II was buffer exchanged into PBS. Then, 50 μl of BiomixA (Avidity), 50 μl of BiomixB (Avidity), and 10 μl of 5 mM D‐biotin (Avidity) were added to the protein along with BirA ligase, followed by overnight incubation at 4°C. The biotinylation was confirmed by Western Blot using Streptavidin HRP‐conjugate (Thermo Scientific). Before use, the reaction mix was buffer‐exchanged into PBS.
BirA‐tagged Sal‐1 DBP‐II was buffer exchanged into PBS. Then, 50 μl of BiomixA (Avidity), 50 μl of BiomixB (Avidity), and 10 μl of 5 mM D‐biotin (Avidity) were added to the protein along with BirA ligase, followed by overnight incubation at 4°C. The biotinylation was confirmed by Western Blot using Streptavidin HRP‐conjugate (Thermo Scientific). Before use, the reaction mix was buffer‐exchanged into PBS.
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!