The purified CRD2 13 (link) was biotinylated using EZ-Link NHS-PEG4-Biotin (Thermo Fisher Scientific) at pH 6.5. The pull-down assays were performed in 1 ml binding buffer, which was composed of 50 mM Tris (pH 7.5) or 20 mM sodium acetate (pH 5.0), together with 400 mM NaCl, 1 mM TCEP, and 0.1% Tween-20. Biotin-labeled CRD2 (~5 µg) was incubated with Strep-Tactin®XT Superflow® resins (IBA Lifesciences) at room temperature for 30 minutes, and the unbound protein was washed away using the binding buffer. The CRD2-bound resins were mixed with a ~2-fold molar excess of TcdB for 30 minutes at room temperature. The resins were then washed twice, and the bound proteins were released from the resins with 50 mM D-biotin and further examined by 4–20% SDS-PAGE.
Ez link nhs peg4 biotin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
EZ-Link NHS-PEG4-Biotin is a water-soluble, sulfhydryl-reactive biotinylation reagent. It provides a spacer arm between the biotin and the target molecule, helping to minimize steric hindrance.
Automatically generated - may contain errors
Lab products found in correlation
Zeba spin desalting column, by Thermo Fisher Scientific (8 mentions)
Octet red96, by Molecular Devices (5 mentions)
Octet red96 device, by Molecular Devices (4 mentions)
Biaevaluation 3, by GE Healthcare (4 mentions)
Octet red, by Molecular Devices (4 mentions)
Data analysis software, by Molecular Devices (4 mentions)
Sensor chip cap, by GE Healthcare (3 mentions)
Pd 10 desalting column, by GE Healthcare (3 mentions)
Streptavidin biosensor pins, by Molecular Devices (3 mentions)
Streptavidin biosensor, by Molecular Devices (3 mentions)
Zeba spin 7k mwco, by Thermo Fisher Scientific (3 mentions)
Adi spp 555, by Enzo Life Sciences (3 mentions)
Strep tactin xt superflow resin, by IBA Lifesciences (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Biotin capture kit, by GE Healthcare (2 mentions)
Biacore t200, by GE Healthcare (2 mentions)
Zeba spin 7 kda molecular weight cut off, by Thermo Fisher Scientific (2 mentions)
Octet system, by Molecular Devices (2 mentions)
Centricon centrifugal filter with a mwco of 10 000, by Merck Group (2 mentions)
Bovine serum albumin powder, by Merck Group (2 mentions)
74 protocols using ez link nhs peg4 biotin
1
CRD2 Protein-Protein Interaction Assay
2
Biotinylation and Purification of Cell Surface Proteins
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Wild type, Pkd1−/− MEFs, or HEK293T cells co-transfected with TRPP2 and wild type PKD1 (PKD1WT) or PKD1S99I were washed 3 times in PBS (pH 8.0), scraped, and re-suspended in 1 ml PBS (pH 8.0). Cell surface proteins were labelled with 1 mM EZ-Link NHS-PEG4-biotin (Thermo Scientific, 21329) for 30 min at room temperature. Biotinylation was terminated by 3 washes with PBS containing 100 mM Glycine, pH 3.0, and lysed in 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl at pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, and 10% sucrose with protease inhibitor cocktail (Roche Applied Science) at 4°C for 30 min. Lysates were collected by centrifugation (18,000 × g, 20 min) and biotin-labelled surface proteins were captured on streptavidin–agarose beads at 4°C by an overnight incubation. Proteins bound to beads were collected by a brief (1 min) centrifugation, and pellets were washed 3 times with lysis buffer for 20 min at 4°C. Biotin-labeled surface proteins were eluted with SDS gel-loading buffer (50 mM Tris-HCl at pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) and analyzed by western blotting.
3
Binding Affinity of KLK4 Inhibitory Peptide
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Example 4
Binding Affinity of KLK4 Inhibitory Peptide
Surface plasmon resonance analysis was performed using BIAcore T200 (GE healthcare) to measure the binding affinity of KLK4 inhibitory peptides by single cycle kinetics. The complementary strand of DNA of the streptavidin conjugate was captured by hybridization to Sensor Chip CAP (GE healthcare) on which the single-stranded DNA was immobilized. Next, approximately 5 RU of KLK4 biotinylated with EZ-Link NHS-PEG4-Biotin (Thermo Fisher Science) was captured at a flow rate of 10 μL/min and immobilized. Thereafter, three-fold serially diluted KLK4 inhibitory peptide (0.08 to 20 nM) with HBS-EP was added as an analyte at a flow rate of 10 μL/min. Analysis was performed using BIAcore T 200 Evaluation software (version 2.0), and kon and koff were calculated using simple one-to-one Langmuir binding model. The equilibrium constant KD was calculated as a koff/kon ratio. By regenerating Sensor Chip CAP using a regeneration buffer attached to Biotin CAPture Kit (GE healthcare) and repeatedly having biotinylated KLK4 captured, multiple KLK4 inhibitory peptides were measured. All of the three measured KLK4 inhibitory peptides showed a KD value of less than 1 nM, showing that their binding is very strong (
4
Binding Affinity Analysis of C6
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Octet RED96 system (FortéBio Biologics by Molecular Devices, San Jose, California) with super streptavidin (SSA) biosensors was used as an additional method for determining the binding affinity of C6. The loading of HMBS to the SSA sensors required biotinylation, which was carried out at room temperature, mixing 1.5 molar excess of N-hydroxysuccinimide (NHS)-ester biotinylation reagent (EZ-Link NHS-PEG4-Biotin; Thermo Fisher Scientific) to protein. After 30 min, excess of biotin was removed using Zeba spin desalting columns (Thermo Fisher Scientific), and the gel filtration buffer was changed to reaction buffer, PBS-P+ (GE Healthcare), supplemented with 5% DMSO. Sensors were loaded with 5 μg/mL of biotinylated HMBS, reaching 6 nm surface thickness. Triplicates for the concentration series of C6 were measured, and double-reference subtraction was applied for data analysis based on the steady-state kinetics with an equilibrium binding signal (Req) using ForteBio Data analysis 9.0. The allosteric sigmoidal curve fitting was performed using GraphPad Prism.
5
Generation of Recombinant SARS-CoV-2 S Antibody
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The SARS-CoV S glycoprotein specific antibody CR3022 was generated recombinantly using previously reported heavy and light variable domain sequences deposited in GenBank under accession numbers DQ168569 and DQ16857011 (link). Antibody variable domain gene sequences were synthesized by IDT and cloned into human IgG1 and human kappa expression vectors as previously described20 (link). Antibodies were produced in Expi293F cells according to the manufacturer’s recommendations by co-transfecting heavy and light chain plasmids at a ratio of 1:1.5. Antibodies were purified using rProtein A Sepharose Fast Flow antibody purification resin (GE Healthcare) and buffer exchanged into PBS before use. Biotinylated versions of CR3022 used in viral neutralization assays were produced by combining the antibody with a 20 molar excess of EZ-Link NHS-PEG4-Biotin (ThermoFisher Scientific) for 1 hour at room temperatures. Reactions were stopped by adding Tris pH 8 to a final concentration of 10 mM. The biotinylated antibody was then buffer exchanged > 1000X into PBS using a 10 kDa protein spin-concentrator (Amicon).
6
Peptide Enrichment for Pancreatic Cancer
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Single Crystal Silicon Wafer was purchased from Western Minmetals (SC) Corporation. Sulfuric acid (98%), hydrogen peroxide (30%), silver nitrate (>99.8%), ammonium fluoride (≥99.99%), ethanol (>99.5%), and (3-aminopropyl) triethoxysilane (APTES, 99%) were purchased from Sigma-Aldrich. EZ-Link™ NHS-PEG4-Biotin was obtained from Thermo Fisher Scientific. Pancreatic cancer cell lines (BxPC3, Panc-1, and AsPC3) were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA). White blood cells (WBCs) were collected from healthy donors. RPMI cell culture medium was obtained from Thermo Fisher Scientific. Custom peptides were ordered from Chinese Peptide Company (Hangzhou, China). Peptide digestion reagents (including ArgC, AspN, chymotrypsin, GluC, LysargiNase, LysC, LysN, pepsin, and trypsin) were obtained from Wako Chemicals (Richmond, VA, USA).
7
Quantification of FABP Proteins in Plasma
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Plasma samples stored at −80 °C were thawed and centrifuged at 10,000× g for 5 min. All specimen analyses were conducted using a Simoa HD-X system (Quanterix, Billerica, MA, USA). Prior to the assay, the samples were diluted with the sample diluent provided in each assay kit or the sample diluent in the Homebrew Assay Development Kit (Quanterix). The diluted samples were then applied to plates and assayed individually. The subsequent measurements were performed according to the manufacturer’s instructions provided in the kit (Quanterix).
To measure FABP2, FABP3, FABP5, and FABP7 levels, a custom assay system was developed using the Homebrew Assay. The assay was conducted according to the protocol provided with the Simoa Homebrew Assay Development Kit (#101354, Quanterix). The reagents necessary for the assay were prepared using Pierce EDC (#A35391; Thermo Scientific, Waltham, MA, USA) and EZ-Link NHS-PEG4-Biotin (#A39259; Thermo Scientific). Capture antibodies were coated onto magnetic beads at a concentration of 0.3 mg/mL, whereas detector antibodies were biotinylated at a 1:40 ratio. These customized reagents enabled the specific detection of FABPs in the samples.
To measure FABP2, FABP3, FABP5, and FABP7 levels, a custom assay system was developed using the Homebrew Assay. The assay was conducted according to the protocol provided with the Simoa Homebrew Assay Development Kit (#101354, Quanterix). The reagents necessary for the assay were prepared using Pierce EDC (#A35391; Thermo Scientific, Waltham, MA, USA) and EZ-Link NHS-PEG4-Biotin (#A39259; Thermo Scientific). Capture antibodies were coated onto magnetic beads at a concentration of 0.3 mg/mL, whereas detector antibodies were biotinylated at a 1:40 ratio. These customized reagents enabled the specific detection of FABPs in the samples.
8
Determination of IL-1α Aptamer Binding Affinity
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For determination of equilibrium binding constants, purified IL-1α protein was incubated at room temperature for 30 min with a 10-fold molar excess of EZ-Link NHS-Peg4-Biotin (ThermoFisher). Free biotin was removed using a YM-3 spin column (Millipore) and the resulting protein concentration was determined using a Micro BCA assay (ThermoFisher). Equilibrium binding constants (Kd values) of modified aptamers were measured in SB18T buffer. Modified aptamers were 5ʹ end labeled using T4 polynucleotide kinase (New England Biolabs) and γ-[32P]ATP (Perkin-Elmer). Radiolabeled aptamers (~ 20,000 CPM, 0.03 nM) were mixed with Il-1α protein at concentrations ranging from 10−7 to 10−12 M and incubated at 37 °C for 40 min. Bound complexes were partitioned using Dynabeads MyOne streptavidin C1 (Life Technologies) and captured on Durapore filter plates (EMD Millipore). The fraction of bound aptamer was quantified with a phosphorimager. To determine binding affinity, data were fit using the equation: and plotted using GraphPad Prism version 7.00.
9
Biotinylation of Extracellular Vesicles
10
Biolayer Interferometry of TarS-polyRboP Binding
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Biolayer interferometry was performed using an Octet Red instrument (FortéBio Inc.) with streptavidin sensors (FortéBio Inc.). TarS was biotinylated using EZ-Link NHS-PEG4-Biotin (Thermo Scientific, USA). Biolayer interferometry was performed at 25°C in a 96-well plate (Greiner Bio-One) and a 200 μL well volume. After a brief equilibration of the sensors in assay buffer (20 mM Hepes pH 7, 500 mM NaCl), full-length or TarS1-349 was loaded onto sensors for 5 minutes at 300 nM followed by the blocking of unbound streptavidin with 15 μg/mL EZ-Link Biocytin (Thermo Scientific) in Superblock Blocking Buffer (Thermo Scientific). Next, a baseline was acquired for 3 minutes followed by the association of TarS for 5 minutes (kon) and dissociation for 15 minutes (koff) in assay buffer (20 mM Hepes pH 7, 500 mM NaCl). Various optimal concentrations of polyRboP (0.31 mM, 0.62 mM, 1.25 mM, 2.5 mM and 5 mM) were titrated with double referencing to rule out non-specific binding to sensors, and the KD was calculated based on kon and koff rates fitted to a heterogeneous ligand model using the FortéBio data analysis software.
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