Intestinal mucosal permeability was measured by fluorescence tracing method. A section of intestine about 15 cm long was chosen and ligated at one end, and then the tracer (EZ-Link NHS-Biotin) (Thermo Fisher Scientific, MA, USA) was diluted to 2 mg/ml and injected into the distal intestinal lumen slowly. A segment of intestine about 5 cm long was taken and fixed in 4% paraformaldehyde solution for 3 h, then washed with phosphate buffer solution 3 times and sliced. The tissue was incubated with streptavidin at room temperature for 30 min. The distribution of the fluorescent tracer in intestinal tissue was observed by a laser confocal microscope.
Ez link nhs biotin
Manufactured by Thermo Fisher Scientific
Sourced in United States
EZ-Link NHS-Biotin is a water-soluble biotinylation reagent used for labeling primary amines on proteins and other biomolecules. It provides a simple, efficient way to introduce biotin moieties onto target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Super streptavidin ssa high binding biosensors, by Molecular Devices (3 mentions)
Octet kinetics buffer, by Molecular Devices (3 mentions)
Proteiniso ni nta resin, by Transgene (2 mentions)
Octet red software, by Molecular Devices (2 mentions)
Octet red96, by Molecular Devices (2 mentions)
Super streptavidin ssa biosensor tips, by Molecular Devices (2 mentions)
Ez link sulfo nhs biotin, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Mesna, by Merck Group (2 mentions)
Recombinant neuraminidase na, by BEI Resources (2 mentions)
N2 neuraminidase na protein with n terminal histidine tag, by BEI Resources (2 mentions)
Recombinant sars cov 2 spike s1 hfc his tagged protein, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated streptavidin, by Merck Group (2 mentions)
Facscalibur, by BD (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Ab69507, by Abcam (1 mentions)
Pac 1 antibody, by BD (1 mentions)
9eg7 antibody, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Accutase cell detachment solution, by Merck Group (1 mentions)
56 protocols using ez link nhs biotin
1
Intestinal Permeability Measurement
2
In Vivo Red Cell Biotin Labeling
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Red cell half-life was studied in vivo using EZ-Link NHS-Biotin (Thermo Fisher Scientific). Mice were injected intravenously with biotin diluted in a 10% DMSO/90% saline solution at a concentration of 30 mg/kg body weight. Tail bleeds were performed on subsequent days and analysed by flow cytometry. 1-2 μl of blood was stained with FITC-Ter119 and Streptavidin-PE, and the percentage of biotin-positive cells was calculated as a fraction of the total biotin-positive cells on day 1. All antibodies were purchased from BD Sciences and flow cytometry was performed on FACSCalibur (BD Sciences).
3
Integrin-Binding Protein Interaction Study
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Flag antibody (SG4110–16, Shanghai Genomics), 6 × his antibody (SG4110–06, Shanghai Genomics), sharpin antibody (ab174545 and ab69507, Abcam), FAK antibody (#3283, CST) and Y-FAK antibodies (#3285, CST) were used for immunoblotting; PAC1 antibody (340535, BD Biosciences), 9EG7 antibody (553715, BD Biosciences) and 7E2 antibody (DSHB) were used for FACS analysis. Plasmid of GST-fibronectin type III repeats 9–11 (GST-Fn-III) was kindly provided by David Calderwood [18 (link)]. The cDNA of full length sharpin was kindly provide by Ivan Dikic [26 (link)], and subcloned into vectors of pET28a, pHis-1, pGST-1 and pGADT7 for different experiments. The CT of integrin α5β1 and integrin αIIbβ3 were subcloned into pGST-1 vector. Kindlin-1 was subcloned into pET31b, pGST-1 and pGBKT7 vectors. To express and purify proteins, the expression vectors were transformed into Rosetta DE3 strain and induced to express proteins with 0.4 mM of IPTG. GST-tagged or his-tagged proteins were purified by Glutathione Sepharpose (GE) and Ni-NTA agarose (Qiagen) respectively, according to the manufactures’ protocols. The purified GST-Fn-III protein was further labeled with biotin (EZ-Link™ NHS-Biotin, Thermo Fisher).
4
HIV Env SOSIP-Specific B Cell Identification
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3–5 days post nucleofection, B cells were washed in PBS and stained in FACS buffer (PBS + 1% FBS) with randomly biotinylated (EZ-Link NHS-Biotin, ThermoFisher), PGT145 purified C108.c03 HIV Env SOSIP (Voss et al., 2017 (link)) APC labeled streptavidin tetramers (Invitrogen, S32362) as previously described (McCoy and Burton, 2017 (link)). Briefly, 2 μg of biotinylated SOSIP (1 mg/ml) was mixed with 0.5 μl of streptavidin (1 mg/ml) in 7.5 μl PBS and incubated 30 min at room temperature (RT). 2 μl of this solution was then incubated for 45 min with 5 × 106 cells in 100 μl FACS buffer. Cells were again washed and single live B cells positive for APC fluorophore were selected using the FACS ARIA III (BD Biosciences) at the TSRI FACS core. Selection gates were made using non-engineered cell controls incubated with the same probe.
5
Quantifying LPL Binding on Cells
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Bovine LPL generously provided by Gunilla Olivecrona (Department of Biomedical Sciences, Umeå University) (40 ). The LPL binding assays were performed using biotin-labeled LPL using the EZ-Link NHS-Biotin (ThermoFisher). Molar ratio of biotin to LPL was determined in a HABA displacement assay (Pierce Biotin Quantitation Kit). Cells were harvested using Accutase cell detachment solution (Millipore) and washed twice with PBS. Cells were incubated for 15 min at 37 °C in serum-free media in the absence or presence of 5 mU/ml each of recombinant heparin lyases I, II, and III. Treated and untreated cells were washed twice with PBS, chilled on ice for 20 min, and incubated with 50 nM biotinylated LPL in 1% FFA-free bovine serum albumine (BSA) supplemented PBS at 4 °C for 1 h. Following the incubation, cells were washed twice in ice-cold PBS and incubated with 0.4 μg/ml Phycoerythrin-Cy5–conjugated Streptavidin in 1% FFA-free BSA supplemented PBS at 4 °C. A set of cells was exposed only to Phycoerythrin-Cy5–conjugated Streptavidin and was used as background control. Cells were washed twice with PBS and analyzed by flow cytometry.
6
Biotinylated Aβ40 Peptide Binding Assay
7
Biotin-labeled pri-let-7a-1 probe binding assay
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Biotin-labeled pri-let-7a-1 probe was generated by in vitro transcription followed by biotin-labeling with EZ-Link NHS-Biotin (ThermoFisher). A total of 1 pmol of biotin-labeled RNA probes was incubated with increasing concentrations of purified recombinant protein expressed in E. coli in reaction buffer (20 mM HEPES-KOH [pH 8.0], 150 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% [w/v] Triton X-100) at 4°C for 1 h. After the reaction, the samples mixed with loading buffer (10 mM Tris-HCl, 3% [w/v] sucrose and dyes) were loaded on 8% (w/v) native gel and run at a constant voltage of 100 V. After electrophoresis, the probe was transferred onto hybond-N+ membrane and detected using Chemiluminescent Nucleic Acid Detection Module Kit (ThermoFisher).
8
Quantifying Netrin-1 via Bio-Layer Interferometry
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Netrin-1 quantitation was performed using a ForteBio Octet K2 system (Sartorius) based on bio-layer interferometry (Do et al. 2008 ). Specifically, an anti-Netrin1-Fab fragment was biotinylated using amine reactive biotin EZ-Link™ NHS-Biotin (Thermo Fisher Scientific). The biotinylated anti-Netrin1-Fab was immobilized on the ForteBio Streptavidin (SA) Biosensors and equilibrated in collection media (DMEM, 5% FBS, 1 μg/mL doxycycline). Next, a standard curve ranging from 1 to 100 μg/mL of Netrin-1ΔC in collection media was established by measuring the initial binding rate over the first 5 s of association. The binding rate was then plotted against the known concentration to achieve a standard curve (Table S1 ). Original HFBR samples were diluted in collection media by a factor of 3 to allow for signal measurements within the given standard curve range. Binding rates of all samples were measured, and protein quantities were then calculated. All measurements were performed for 80 s at 25 °C and 1000 rpm shake speed using the initial rate over 5 s for analysis. Data were processed with the ForteBio Octet Data Analysis HT software.
9
UV-Mediated Biotin Functionalization
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The coated slides were exposed to 365-nm UV light (2.5 J/cm2) through a custom photomask with an array of 40-μm hexagons (CAD/Art Services) using UV-KUB (Kloe) (fig. S2). The saturating UV dose fully deprotected surface amines inside hexagons. Surface amines located between hexagons were 53% deprotected due to leakiness of the mask (fig. S2). Biotin N-hydroxysuccinimidyl ester (EZ-link NHS-Biotin, Thermo Scientific) dissolved in borate buffer to a final concentration of 0.5 mg/ml was applied to the surface for 40 min and reacted with exposed amines. The slides were then washed with water and dried.
10
Mosquito Biotin-Labeling Protein Purification
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A. aegypti was maintained in laboratory and reared under optimal conditions of 28 °C and 70–80% relative humidity with a 14 h light and 10 h dark photoperiod. The recombinant Bt strain pCG6 producing Cry11Aa and with high toxicity against mosquitoes was provided by Dr. Sarjeet R. Gill laboratory at University of California, Riverside, CA, USA. Escherichia coli (E. coli) strain DH5α and BL21 (DE3) were purchased from TaKaRa (Dalian, China). Plasmid pMD18-T (TaKaRa, Dalian, China) and pET32a vector used in this study were stored in our lab. EZ-Link-NHS-Biotin was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Expression vectors for recombinant A. aegypti ALP1 were obtained from Dr. Sarjeet R. Gill laboratory. Biotin specific antibody Streptavidin/AP was purchased from BIOSS antibodies (Beijing, China). Streptavidin horse-radish peroxidises (HRP) conjugate antibody was purchased from Beyotime biotech (Shanghai, China), Anti-6xHis antibody and rabbit polyclonal antibodies were from BBI life science (Shanghai, China). ProteinIso® Ni-NTA resins were obtained from TransGen Biotech (Beijing, China).
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