The binding studies were carried out using the ProteOn XPR36 Protein Interaction Assay V.3.1 from Bio-Rad. The GLM sensor chip was activated by reaction with EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) and sulfo-NHS (N-hydroxysulfosuccinimide) (Sigma). Following this, 10 μg/ml of various bnAbs (VRC01, 2G12, PGDM1400, PGT145, PG9, PGT151,PGT128) and non-nAbs (17b, b6) were coupled in the presence of 10 mM sodium acetate buffer pH 4.5 at 30 μl/min for 100 s in various channels, leaving one channel as blank that acts as a reference. The Response Units for coupling were monitored till ~1500–2000RU was immobilized. Finally, the excess sulfo-NHS esters were quenched using 1M ethanolamine. NFL Wt, D1, D2 and D3 were passed at a flow rate of 30 μl/min for 300 s over the chip surface, followed by a dissociation step of 600 s. A lane without any immobilization but blocked with ethanolamine was also used to monitor non-specific binding. After each kinetic assay, the chip was regenerated in 4M MgCl2. Various concentrations of the NFL Wt, D1, D2 and D3 (250 nM, 125 nM, 62.2 nM, 31.1 nM, 15.6 nM) in 1X PBST were used for binding studies. The kinetic parameters were obtained by fitting the data to the simple 1:1 Langmuir interaction model using Proteon Manager.
Proteon xpr36 protein interaction assay v 3
Manufactured by Bio-Rad
The ProteOn XPR36 Protein Interaction Assay V.3.1 is a label-free, real-time biomolecular interaction analysis system. It enables the simultaneous measurement of up to 36 interactions between a set of analytes and ligands in a single experiment.
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Lab products found in correlation
3 protocols using proteon xpr36 protein interaction assay v 3
1
Binding Kinetics of HIV-1 Envelope Proteins
2
Kinetic Analysis of BCL-2 Binding
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Binding studies were performed using ProteOn XPR36 Protein Interaction Assay V.3.1 from Bio-Rad. Upon GLM chip activation with EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) and sulfo-NHS (N-hydroxysulfosuccinimide) (Sigma, St. Louis, MO, USA), the BCL-2 protein (Sigma) was immobilized using a 10 mM sodium acetate buffer pH 4.0 at 30 μL/min for 100 s in various channels except for the blank channel. The response units for immobilization were monitored until ~4000 RU followed by quenching the excess with 1 M of ethanolamine. At a flow rate of 20 μL/min, the compounds were passed onto the BCL-2 immobilized chip surface for 300 s followed by dissociation for 600 s. The kinetics of the association and dissociation were analyzed and fitted by a 1:1 Langmuir interaction model using ProteOn Manager. The time scale analysis was performed by pre-incubating the BCL-2 immobilized chip with SM396 and the kinetics were studied at 0 h, 3 h, and 6 h time points.
3
Binding Studies of HIV-1 Neutralizing Antibodies
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The binding studies were carried out using the ProteOn XPR36 Protein Interaction Assay V.3.1 from Bio-Rad. The GLM sensor chip was activated by reaction with EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) and sulfo-NHS (N-hydroxysulfosuccinimide) (Sigma). Following this, 10 μg/ml of various bnAbs (VRC01, 2G12, PGDM1400, PGT145, PG9, PGT151,PGT128) and non-nAbs (17b, b6) were coupled in the presence of 10 mM sodium acetate buffer pH 4.5 at 30 μl/min for 100 s in various channels, leaving one channel as blank that acts as a reference. The Response Units for coupling were monitored till ~1500-2000RU was immobilized. Finally, the excess sulfo-NHS esters were quenched using 1M ethanolamine. NFL Wt, D1, D2 and D3 were passed at a flow rate of 30 μl/min for 300 s over the chip surface, followed by a dissociation step of 600 s. A lane without any immobilization but blocked with ethanolamine was also used to monitor non-specific binding. After each kinetic assay, the chip was regenerated in 4M MgCl2. Various concentrations of the NFL Wt, D1, D2 and D3 (250 nM, 125 nM, 62.2 nM, 31.1 nM, 15.6 nM) in 1X PBST were used for binding studies. The kinetic parameters were obtained by fitting the data to the simple 1:1 Langmuir interaction model using Proteon Manager.
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