The alkaline phosphatase (ALP) of BBI (CAT: 03535452) and homemade ACE2 (Fc-tag, expressed by human 293 cells) were used for conjugating. ALP and ACE2 were dialyzed in 0.05 M MES, pH 4.5, mixed at a molar ratio of 2 ALP to 1 ACE2, and then EDC and sulfo-NHS (Thermo Fisher) were added to obtain a concentration of 2 mM EDC and 5 mM sulfo-NHS, reacted for 2 h at room temperature. The conjugate was blocked by glycine and purified by gel filtration, stored at 2–8 °C.
Sulfo nhs
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sulfo-NHS is a water-soluble, amine-reactive compound commonly used in protein conjugation and modification reactions. It functions as an activated ester, facilitating the formation of stable amide bonds between primary amines and carboxyl groups. The core purpose of Sulfo-NHS is to enable efficient protein labeling and cross-linking in various biochemical and analytical applications.
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Lab products found in correlation
Forecyt, by Intellicyt (4 mentions)
Lyophilized guinea pig complement, by Cedarlane (3 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride edc, by Thermo Fisher Scientific (2 mentions)
Calcium and magnesium, by Boston BioProducts (2 mentions)
Red neutravidin conjugated microspheres, by Thermo Fisher Scientific (2 mentions)
Carboxylate modified microspheres, by Thermo Fisher Scientific (2 mentions)
Guinea pig complement ce, by MP Biomedicals (2 mentions)
Neutravidin, by Thermo Fisher Scientific (2 mentions)
Automated plate washer, by Tecan (2 mentions)
Sulfo nhs, by Merck Group (2 mentions)
Neutravidin conjugated beads, by Thermo Fisher Scientific (2 mentions)
C18 chromatography, by Waters Corporation (2 mentions)
Ique screener plus, by Intellicyt (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Heparin sodium, by Merck Group (1 mentions)
Nicolet avatar 360, by Thermo Fisher Scientific (1 mentions)
Bodipy fl eda, by Thermo Fisher Scientific (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Chitosan, by Merck Group (1 mentions)
59 protocols using sulfo nhs
1
Conjugation of Alkaline Phosphatase and ACE2
2
LcrV Conjugation to Fluorescent Particles
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Conjugation of LcrV to amine-coated, fluorescent Nile blue particles (Spherotech) was performed using EDC and Sulfo-NHS (ThermoFisher) by a two-step procedure [19 (link),20 ]. Briefly, purified LcrV at a concentration of 1 mg/ml was activated with EDC and sulfo NHS (Thermo) in MES buffer, pH = 6.0 for 30. After quenching excess EDC with β-mercaptoethanol (Sigma), the fluorescent particles were added in carbonate buffer, pH = 8.3. The LcrV conjugated particles were then washed three times in PBS and stored at 4°C.
3
Bead-Based Coupling of Recombinant MAP Proteins
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A total of 100 μg of each purified recombinant MAP protein was coupled to fluorescent beads (Luminex, Austin, TX) at room temperature according to the manufacturer’s instructions. MAP1272c was coupled to bead 33, MAP1569 to 34, MAP2121c to 35, MAP2942c to 36, MAP2609 to 37, and MAP1201c+2942c to 38. All centrifugation steps were performed at 14,000 x g for 4 minutes (min). In brief, the beads were resuspended by vortexing and sonication for 20 seconds. For activation, 5x106 beads were washed once in deionized H2O. Beads were resuspended in 80 μl of 100 mM sodium phosphate buffer, pH 6.2 and 10 μl of Sulfo-NHS (50 mg/ml,) and 10 μl 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, 50 mg/ml, both from Pierce Biotechnology Inc., Rockford, IL) were added and incubated for 20 min. The beads were then washed twice with 50 mM 2-[N-morpholino] ethanesulfonic acid pH 5.0 (MES) and resuspended in MES solution. These activated beads were used for MAP antigen coupling using 100 μg of each antigen. The coupling of the MAP antigens was performed for three hours with rotation. After coupling, the beads were resuspended in blocking buffer (PBS with 1% (w/v) BSA and 0.05% (w/v) sodium azide) and incubated for 30 min. The beads were washed three time in PBS with 0.1% (w/v) BSA, 0.02% (v/v) Tween 20 and 0.05% (w/v) sodium azide (PBS-T), counted and stored in the dark at 2–8°C.
4
Aminolysis-Heparin Functionalization of PCU Grafts
5
Fluorescent Labeling of Antifungal Protein
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PeAfpA was labelled with the green fluorophore BODIPY™-FL EDA (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine, Invitrogen, Thermo Fisher Scientific) as described with minor changes (Sonderegger et al. 2017 (link)). In brief, 3-mg lyophilized PeAfpA were dissolved in 1 mL of 0.1 M MES (2-(N-morpholino)ethanesulfonic acid) buffer pH 4.5. Subsequently, a reaction mixture containing 200 μL of PeAfpA, 30 µL of 0.1 M EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, Invitrogen), 15 µL of 100 mM Sulfo-NHS (N-hydroxysulfosuccinimide, Invitrogen) and 30 µL of 50 mM BODIPY in MES buffer was incubated in darkness at 25 °C, with gently mixing for 3 h. The reaction mixture was dialyzed (3.5 kDa molecular weight cut-off; Thermo Scientific, Thermo Fisher Scientific) several times against deionized water at 4 °C to remove free BODIPY and salts. Labelling efficiency and protein concentration were determined spectrophotometrically. Labelled protein retained full antifungal activity (Supplemental Fig. S1 ).
6
PAFC Labeling and Antifungal Activity
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For visualization in uptake studies PAFC was labeled with the green fluorophore BODIPY™ FL EDA (Bd) (Invitrogen, Carlsbad, CA, USA) as described previously [27 (link)]. In brief, 0.4 mM PAFC was labeled with 10 mM Bd in the presence of 10 mM EDAC and 5 mM Sulfo-NHS (Invitrogen, Carlsbad, CA, USA) in 100 M MES-buffer (pH 4.5). The reaction mixture was incubated with continuous shaking at 200 rpm overnight at 25 °C in the dark. The labeled AMP (PAFC-Bd) was dialyzed against ddH2O to remove excess Bd and concentrated using Amicon®Ultra centrifugal filters (3 kDa MWCO; Merck Millipore, Burlington, MA, USA). The antifungal activity of PAFC-Bd was tested by broth microdilution assay as described above.
7
Folate-Functionalized SWCNTs for Targeted Delivery
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CVD SWCNTs (10 mg) (Xianfeng Co.) were sonicated in a solution containing 30 ml H2SO4 (98%) and 10 ml HNO3 (65%) for 24 h. The material was washed and dissolved in chitosan (CS, M = 4000–6000, Sigma-Aldrich) solution (1:2 w/w) with sonication. The mixture was centrifuged at 15,000 g for 1 h to remove aggregates, and the supernatant was dialyzed through a 10 kDa molecular-weight cut-off (MWCO) membrane (Millipore Amicon). The concentration of CS/SWCNTs was determined by UV-vis-NIR spectroscopy. For conjugation of folate, 100 μl of folate (1 mM in PBS) was added to 10 mL CS/SWCNTs solution (1 mg ml−1), and then 50 μl of 1 mM 1-ethyl-3-(3-dimethylaminopropy) carbodiimide hydrochloride (EDC, Invitrogen) and 1 mM N -hydroxysulfosuccinimide (sulfo-NHS, Invitrogen) were added. For fluorescent labeling, 100 μl of rhodamine-6G (100 μM in PBS) were added to the FA-SWCNTs together with 10 μl EDC and 10 μl sulfo-NHS and incubated for 24 h. The final solution was dialyzed three times to remove unconjugated molecules.
8
ADCD Assay for Complement Activation
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ADCD was conducted as previously described58 (link). Briefly, NVX-CoV2373 Spike protein was biotinylated using EDC (Thermo Fisher) and Sulfo-NHS (Thermo Fisher), and then coupled to red Neutravidin-conjugated microspheres (Thermo Fisher) or directly coupled to Carboxylate-Modified microspheres (Thermo Fisher). Immune complexes were formed by incubating the bead + protein conjugates with diluted serum for 2 hours at 37°C, and then washed to remove unbound antibody. The immune complexes were then incubated with lyophilized guinea pig complement (Cedarlane) and diluted in gelatin veronal buffer with calcium and magnesium (Boston Bioproducts) for 30 minutes. C3 bound to immune complexes was detected by fluorescein-conjugated goat IgG fraction to guinea pig Complement Ce (MP Biomedicals). Flow cytometry was performed to identify the percentage of beads with bound C3. Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1).
9
Immuno-Fluorescent Labeling of Extracellular Vesicles
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FLEX chips were serially cleaned with acetone, IPA, and deionized water. The sensor chip surface was functionalized with a mixture of SH‐PEG‐COOH 1 k (Nanocs) and SH‐mPEG 0.35 k at a ratio of 1:3 overnight. For EV capture, the gold surface was incubated in 0.2 m EDC (Thermo) and 0.05 m sulfo‐NHS (Thermo) for 7 min to capture EVs by covalent bonding. After gently washing with PBS, EVs were introduced to the sensor chip and incubated for 30 min. After EV capture, EVs were fixed by 4% paraformaldehyde for 10 min, followed by blocking with 2% BSA for 20 min. The captured EV were immuno‐fluorescently labeled by primary antibodies for 60 min, followed by secondary antibody (AlexaFluor 647 anti‐mouse) incubation for 30 min. Antibodies were diluted in 0.2% BSA solution, and staining was performed under agitation. Each antibody was diluted with its dilution factor, which was determined by screening optimal antibody concentrations (CD63:1/100, EpCAM:1/20, MUC1:1/800, EGFR:1:20). Finally, the chips were mounted with a mounting solution (ProLong Gold Antifade mountant, Thermo Fisher) and covered with a glass coverslip. Fluorescence images were acquired on Nikon Ti inverted automated epifluorescence microscope with a 40 × (NA = 0.95) objective lens.
10
Isotyping and FcR Profiling of Spike Antigens
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Isotyping and FcR profiling was conducted as previously described60 (link),61 (link). Briefly, antigens (NVX-CoV2373 Spike, SARS-CoV-2 Spike, S1, RBD, S2, HKU-1 RBD, or OC43 RBD) were carboxyl coupled to magnetic Luminex microplex carboxylated beads (Luminex Corporation) using NHS-ester linkages with Sulfo-NHS and EDC (Thermo Fisher), and then incubated with serum for 2 hours at 37°C. Isotyping was performed by incubating the immune complexes with secondary mouse-anti-rhesus antibody detectors for each isotype (IgG1, IgG2, IgG3, IgG4, IgA), then detected with tertiary anti-mouse-IgG antibodies conjugated to PE. FcR binding was quantified by incubating immune complexes with biotinylated FcRs (FcγR2A-1, FcγR2A-2, FcγR3A, courtesy of Duke Protein Production Facility) conjugated to Steptavidin-PE (Prozyme). Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1).
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