Carboxylated microspheres (5x106, Luminex, Cat. No. MC10043) were coupled with 25 μg of gp70-V1V2 scaffolds by covalent N-hydroxysulfosuccimide-ester linkages using a combination of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) and N-hydroxysulfosuccimide (ThermoScientific) in phosphate buffered saline (PBS), pH 7.4 according to the manufacturer’s instructions. C3d complement deposition on gp70-V1V2 coated beads was performed in 96 well black plates (Bio-Rad, Cat. No. 171025001). To obtain a final dilution of 1:30, samples and NHS were separately diluted 1:7.5 in PBS and 25 μl of each were added to duplicate wells containing 2,500 antigen-coated beads in 50 μl of PBS. After incubation at room temperature for 1 hour the beads were washed with Bioplex buffer (BioRad, Cat. No. 171304500) and incubated with 100 μl of a 1:500 dilution of a biotinylated mouse monoclonal antibody to human C3d (Quidel, Cat. No. A702) at room temperature for 30 minutes. The beads were washed with Bioplex buffer and incubated at room temperature with 100 μl of a 1:500 dilution of PE-Streptavidin (BD Pharmingen, Cat. No. 554061) for 30 minutes. After a final wash with Bioplex buffer, beads were resuspended in 100 μl of Bioplex buffer and mean fluorescence intensity (MFI) was determined using MAGPIX fluorescence imagery (Luminex).
1 ethyl 3 3 dimethylaminopropyl carbodiimide hcl
Manufactured by Thermo Fisher Scientific
Sourced in United States
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl is a chemical compound commonly used as a coupling agent in various biochemical applications. It is a water-soluble, odorless, and crystalline solid that can facilitate the formation of amide bonds between carboxylic acids and primary amines.
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Lab products found in correlation
N hydroxysulfosuccimide, by Thermo Fisher Scientific (1 mentions)
Streptavidin pe, by BD (1 mentions)
Hydroxybenzotriazole, by Merck Group (1 mentions)
Sodium phosphate monobasic anhydrous, by Thermo Fisher Scientific (1 mentions)
Saline sodium citrate buffer, by Merck Group (1 mentions)
N n diisopropylcarbodiimide, by Merck Group (1 mentions)
2 hydroxy 2 methylpropiophenone darocur 1173, by Merck Group (1 mentions)
N boc ethylenediamine, by Merck Group (1 mentions)
Tween 20, by Thermo Fisher Scientific (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Tetrahydrofuran, by Merck Group (1 mentions)
N hydroxysuccinimide, by Thermo Fisher Scientific (1 mentions)
2 propanol, by Thermo Fisher Scientific (1 mentions)
Hexadecane, by Thermo Fisher Scientific (1 mentions)
Sodium phosphate dibasic anhydrous, by Thermo Fisher Scientific (1 mentions)
12 mercaptododecanoic acid, by Merck Group (1 mentions)
N hydroxysulfosuccinimide, by Thermo Fisher Scientific (1 mentions)
4 protocols using 1 ethyl 3 3 dimethylaminopropyl carbodiimide hcl
1
C3d Complement Deposition Assay
2
Biomolecule Immobilization Protocol
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(AAm) (99.9%), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide HCl (EDC), N-hydroxysuccinimide (NHS), 2-(4morpholino)ethanesulfonic acid (MES), hexadecane, 2-propanol, sodium phosphate monobasic anhydrous (99%), sodium phosphate dibasic anhydrous (≥ 99%), Tween 20, poly(dimethylsiloxane) (PDMS) elastomer kits (Sylgard 184) and sodium hydroxide (NaOH) were purchased from Thermo Fisher Scientific (Waltham, MA). Acrylic acid (AAc) anhydrous (180-200ppm MEHQ inhibitor,99%), 2-hydroxy-2-methylpropiophenone (Darocur 1173, photoinitiator), saline sodium citrate (SSC) buffer (20× concentrate, molecular biology grade), N-Boc ethylene diamine, hydroxybenzotriazole (HOBt), N,N-dimethylformamide (DMF), Tetrahydrofuran (THF), N,N'-Diisopropylcarbodiimide (DIC), cis-dichlorobis(2,2'-bipyridine)ruthenium(II), N,N'-Methylenebis(acrylamide) (Bis) were purchased from Sigma Aldrich.
4-Methyl-4'carboxy-2,2'-bipyridine was purchased from Carbosynth. All chemicals were of analytical grade and used without further purification.
4-Methyl-4'carboxy-2,2'-bipyridine was purchased from Carbosynth. All chemicals were of analytical grade and used without further purification.
3
Synthesis and Functionalization of Gold Nanoparticles
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The linker MDDA (12-Mercaptododecanoic acid, Sigma-Aldrich) conjugates between the GNP and the barbiturate. One side of the MDDA chain (thiol) connects to the gold via semi-covalent bonding, while the other side of the MDDA chain, carboxylic acid, binds to the negatively charged oxygen of the barbiturate. To each solution of the various GNP sizes, MDDA was added in excessive amounts, and the mixture was stirred for another four hours. Following this step, the solutions were centrifuged in order to reach higher concentrations. Next, the activating agents EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl, Thermo Scientific) and NHS (N-Hydroxysulfosuccinimide sodium salt, Chem-Impex International) were added to the mixture together with the barbiturate, and stirred overnight (Figure 8 ). The solutions were centrifuged again in order to increase concentration.![]()
4
Protein Conjugation on Carboxyl Beads
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LEGENDplex carboxyl beads containing allophycocyanin (APC) fluorescence (BioLegend, USA) were used for the assay development. The surface chemistry of these beads allows for the covalent coupling of different antigens. Antigen conjugation to these beads can be detected using a secondary antibody coupled to a fluorochrome other than APC. The bead conjugation was carried out according to the manufacturer's recommendations.
Briefly, LEGENDplex carboxyl beads were washed and resuspended in coupling buffer, then activated by incubation with N-hydroxysulfosuccinimide (Thermo Fisher Scientific, USA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (Thermo Fisher Scientific) for 20 min at room temperature.
After activation, the beads were rewashed and resuspended in a coupling buffer. Zika E proteins were later conjugated to activated carboxyl beads by vortexing and incubation at room temperature for 2 h. Conjugated beads were then blocked with blocking buffer and used for flow cytometric assays. The protein conjugation and the specificity of these beads were confirmed by staining with anti-E (BEI Resources, USA) and anti-NS-1 (Thermo Fisher Scientific) antibodies. Protein conjugated beads were first stained with primary antibodies for 45 min at room temperature and then washed with staining buffer (PBS
Briefly, LEGENDplex carboxyl beads were washed and resuspended in coupling buffer, then activated by incubation with N-hydroxysulfosuccinimide (Thermo Fisher Scientific, USA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (Thermo Fisher Scientific) for 20 min at room temperature.
After activation, the beads were rewashed and resuspended in a coupling buffer. Zika E proteins were later conjugated to activated carboxyl beads by vortexing and incubation at room temperature for 2 h. Conjugated beads were then blocked with blocking buffer and used for flow cytometric assays. The protein conjugation and the specificity of these beads were confirmed by staining with anti-E (BEI Resources, USA) and anti-NS-1 (Thermo Fisher Scientific) antibodies. Protein conjugated beads were first stained with primary antibodies for 45 min at room temperature and then washed with staining buffer (PBS
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