SPR instrumentation (XPR36), GLC sensor chip, and amine coupling reagents containing N-hydroxysulfosuccinimide (sulfo-NHS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDAC), and ethanolamine HCl were obtained from Bio-Rad. Synthetic refolded Agaphelin was diluted to a concentration of 100 µg/ml in NaOAc buffer (10 mM, pH 4.5) and coupled to the surface of a GLC chip using the manufacture's amine-coupling chemistry as described in the XPR36 system manual. Briefly, the surface of the sensor chip was first activated with EDC/NHS, followed by addition of the peptide. The surface was blocked using ethanolamine. Employing these conditions, surfaces containing densities of 408.27 resonance units of peptide were generated. Surface regeneration was done using 10 mM HCl. Sensograms were recorded and normalized to a baseline of 0 resonance units. Equivalent volumes of elastase were also injected over a mock, no-protein, blocked surface to serve as a blank sensogram for subtraction of bulk refractive index background. Sensograms were analyzed for fitting using XPR36 software (Bio-Rad). A Langmuir single-site binding model (A+B = AB) was used for analysis of interaction of the Kazal peptide and elastase surface.
Sulfo nhs
Manufactured by Bio-Rad
Sourced in Australia
Sulfo-NHS is a water-soluble, amine-reactive reagent used for protein modification and conjugation. It functions as an N-hydroxysulfosuccinimide ester, enabling the formation of stable amide bonds between the reagent and primary amino groups present in proteins and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Biacore t200, by GE Healthcare (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Ethanolamine hcl, by Bio-Rad (1 mentions)
Bio plex cooh beads, by Bio-Rad (1 mentions)
Sulfo nhs, by Thermo Fisher Scientific (1 mentions)
Sodium acetate, by ITW Reagents (1 mentions)
Sodium chloride (nacl), by Helicon (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by ITW Reagents (1 mentions)
Ifn β, by Merck Group (1 mentions)
Dithiothreitol (dtt), by ITW Reagents (1 mentions)
Nap 5 column, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by ITW Reagents (1 mentions)
Hepes, by ITW Reagents (1 mentions)
Tween 20, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Cacl2, by Merck Group (1 mentions)
Mcf7 cell line, by European Collection of Authenticated Cell Cultures (1 mentions)
Estradiol, by Merck Group (1 mentions)
5 protocols using sulfo nhs
1
Kinetic Analysis of Agaphelin-Elastase Binding
2
Coupling Monoclonal IgM Antibodies to Polystyrene Beads
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monoclonal IgM (MPFM-55A, Abcam; ab9206) antibodies were covalently bound to polystyrene BioPlex® COOH beads (BioRad; 1715060XX) by the commonly-used EDC/Sulfo-NHS intermediate reaction. Reactive esters were formed on the carboxylated beads in the presence of EDAC [1-Ethyl-3-(3ʹ-dimethylaminopropyl)carbodiimide](EMD Millipore; 341006) and Sulfo-NHS [N-hydroxysulfosuccinimide] (ThermoScientific; 24510) under light agitation for 20 min. Carboxyl to antibody amine crosslinking took place in the presence of activation buffer (LuminexCorp; 11–25171) under light agitation for 2 h. Nonspecific protein binding was blocked by BSA incubation (PBS pH7.2, 0.05% Tween20 [PBS-T] + 1% BSA) for 30 min and beads resuspended in blocking buffer with the addition of 0.02% NaN3. Once the optimal coupling concentration of capture antibody to beads was found (S1 Fig ), a large-scale bead coupling was done to cover all experiments.
3
Recombinant Protein Characterization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human interferon beta-1a produced in CHO-K1 cells (IFN-β) was purchased from Merck (Rebif®). Human S100B was expressed in E. coli and purified as described in ref. [62 (link)]. Protein concentrations were measured spectrophotometrically according to ref. [64 (link)].
Sodium acetate, HEPES, NaOH, DTT and SDS were from PanReac AppliChem. Sodium chloride was from Helicon (Moscow, Russia). CaCl2, EDTA and TWEEN 20 were purchased from Sigma-Aldrich Co. NAP-5 column was from Cytiva.
ProteOn™ GLH sensor chip, amine coupling kit, EDAC and sulfo-NHS were from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).
MCF-7 cell line was from European Collection of Authenticated Cell Cultures. Crystal violet was from Sigma-Aldrich Co.
Sodium acetate, HEPES, NaOH, DTT and SDS were from PanReac AppliChem. Sodium chloride was from Helicon (Moscow, Russia). CaCl2, EDTA and TWEEN 20 were purchased from Sigma-Aldrich Co. NAP-5 column was from Cytiva.
ProteOn™ GLH sensor chip, amine coupling kit, EDAC and sulfo-NHS were from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).
MCF-7 cell line was from European Collection of Authenticated Cell Cultures. Crystal violet was from Sigma-Aldrich Co.
4
SPR Analysis of Estradiol Analogues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Commercially available reagents were used without additional purification unless otherwise stated. estradiol, estradiol 3-sulfate sodium salt, estradiol 3,17-disulfate dipotassium salt, estrone sulfate potassium salt were purchased from Sigma-Aldrich (Sydney, Australia). Methanol and dimethyl sulfoxide (DMSO) were purchased from Thermo Fisher Scientific (Scoresby, Australia). PBST (phosphate buffered saline, pH 7.4 with 0.05% Tween 20) was purchased from Sigma, and SPR reagents (40 mM EDC, 10 mM sulfo-NHS, 10 mM sodium acetate, 1 M ethanolamine HCl, pH 8.5) were purchased from Bio-Rad Laboratories (Gladesville, Australia).
SPR binding affinity measurements were performed using the Biacore T200 with a CM5 sensor chip (GE Healthcare Life Sciences, Sydney, Australia). The experiment was prepared and obtained with the Biacore T200 Control Software, and analysed with the Biacore T200 Evaluation Software.
GraphPad Prism v7.02 software59 was used to develop a nonlinear regression fit of the data collected in the inhibition assays. The IC50 is reported with the standard error.
SPR binding affinity measurements were performed using the Biacore T200 with a CM5 sensor chip (GE Healthcare Life Sciences, Sydney, Australia). The experiment was prepared and obtained with the Biacore T200 Control Software, and analysed with the Biacore T200 Evaluation Software.
GraphPad Prism v7.02 software59 was used to develop a nonlinear regression fit of the data collected in the inhibition assays. The IC50 is reported with the standard error.
5
Surface Plasmon Resonance Screening
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Commercially available reagents were used without additional purification unless otherwise stated. The fragment library (1000 wells, each containing 1 mg of a fragment compound) was purchased from Life Chemicals (Ontario, Canada). Phosphate-buffered saline (PBST; pH 7.4 with 0.05% Tween 20) was purchased from Sigma-Aldrich (Sydney, Australia). SPR reagents (40 mM EDC, 10 mM sulfo-NHS, 10 mM sodium acetate, 1 M ethanolamine HCl, pH 8.5) were purchased from Bio-Rad Laboratories (Gladesville, Australia). Methanol and DMSO were purchased from Thermo Fisher Scientific (Scoresby, Australia).
The SPR screening protocol was prepared with the Biacore T200 control software, measurements were performed using the Biacore T200 with a CM5 sensor chip, and the results were analyzed with the Biacore T200 evaluation software (GE Healthcare Life Sciences, Sydney, Australia).
The IC 50 , reported with standard error, was determined using GraphPad Prism v7.02 software (GraphPad, La Jolla, CA), by developing a nonlinear regression fit of the data collected from the inhibition assays. Images of docked compounds were generated by the PyMOL Molecular Graphics System (Schrodinger LLC, Cambridge, MA).
The SPR screening protocol was prepared with the Biacore T200 control software, measurements were performed using the Biacore T200 with a CM5 sensor chip, and the results were analyzed with the Biacore T200 evaluation software (GE Healthcare Life Sciences, Sydney, Australia).
The IC 50 , reported with standard error, was determined using GraphPad Prism v7.02 software (GraphPad, La Jolla, CA), by developing a nonlinear regression fit of the data collected from the inhibition assays. Images of docked compounds were generated by the PyMOL Molecular Graphics System (Schrodinger LLC, Cambridge, MA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!