Biacore T200 (GE Healthcare, USA) surface plasmon resonance (SPR) was utilised to determine the binding affinity between various glycoforms of rituximab and FcγRIIIa-V158. To capture a variety of rituximab glycoforms, a standard primary amine coupling chemistry at pH4.5 was utilised to immobilise protein A on a CM5 biosensor chip (GE Healthcare, USA). A flow cell prepared with BSA was used as reference. Each rituximab glycoform in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05 % surfactant P20) was injected individually at 10 μl/min. The antibodies were captured at 200 RU for FcγRIIIa-V158. 2 × serial dilutions of FcγRIIIa receptor in HBS-P buffer was injected at 30 μl/min. After each cycle, the surface was regenerated by injecting glycine HCl buffer (10 mM, pH 2.0). To obtain the equilibrium constant KD, the experimental data was fitted into a 1:1 Langmuir binding model utilising the Biacore T200 evaluation software (GE Healthcare).
Biacore t200
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom, Australia, Germany, Georgia, France, Japan, New Zealand
The Biacore T200 is a label-free, real-time interaction analysis system designed for studying molecular interactions. It provides quantitative data on binding kinetics, affinity, and specificity between molecules. The system utilizes surface plasmon resonance (SPR) technology to detect and measure these interactions without the need for labeling.
Automatically generated - may contain errors
Lab products found in correlation
Cm5 sensor chip, by GE Healthcare (76 mentions)
Biaevaluation software, by GE Healthcare (50 mentions)
Cm5 chip, by GE Healthcare (50 mentions)
Biacore t200 evaluation software, by GE Healthcare (39 mentions)
Amine coupling kit, by GE Healthcare (29 mentions)
Biacore evaluation software, by GE Healthcare (15 mentions)
Hbs ep buffer, by GE Healthcare (14 mentions)
Series s sensor chip cm5, by GE Healthcare (14 mentions)
Hbs ep, by GE Healthcare (12 mentions)
Series s sensor chip sa, by GE Healthcare (10 mentions)
Sensor chip sa, by GE Healthcare (10 mentions)
Biacore t200 evaluation software 3, by GE Healthcare (9 mentions)
Nta sensor chip, by GE Healthcare (9 mentions)
Surfactant p20, by GE Healthcare (8 mentions)
Biotin capture kit, by GE Healthcare (6 mentions)
Pbs p, by GE Healthcare (6 mentions)
Gst capture kit, by GE Healthcare (6 mentions)
Origin 8, by OriginLab (6 mentions)
Biacore 2000, by GE Healthcare (5 mentions)
Cm5 sensor chip, by Cytiva (5 mentions)
707 protocols using biacore t200
1
Rituximab Glycoform-FcγRIIIa Affinity
2
Cas9 Binding Kinetics via SPR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SPR binding assays were performed using a CM5 chip on a Biacore T200 (GE Healthcare). The amine coupling ligand immobilization procedure was performed at a flow rate of 5 μl/min. The CM5 chip was activated with a mixture of 0.1 M N-hydroxysuccinimide and 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride at a ratio of 1:1 for 400 s. Subsequently, 60 μg/ml of Cas9 dissolved in 10 mM sodium acetate (pH 5.0) was injected for 30 s. The remaining activated carboxyl groups on the sensor chip surface were deactivated with 1 M ethanolamine (pH 8.5) for 400 s. The multi-cycle analysis was performed at a flow rate of 30 μl/min. Each concentration of Csn2 in running buffer (150 mM NaCl, 10 mM Tris–HCl (pH 8.0)) was injected for 600 s, followed by dissociation for 1200 s in a separate analysis cycle. The sensor chip surface was regenerated with 10 mM NaOH between cycles. Data were fit with the bivalent analyte model. The equilibrium dissociation constant (Kd) was determined based on kinetic rate constants calculated using Biacore T200 evaluation software (GE Healthcare).
3
Notch1 Immobilization and SPR Analysis
4
Sitagliptin-Target Protein Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To verify the affinity and kinetics between sitagliptin and the novel target, we performed this experiment using Biacore T200 (GE Healthcare) instrument based on SPR, and used GraphPad Prism 8.0 software to visualize the data results (18 (link), 19 (link)).
Firstly, the amino acid sequence of the target protein was searched by Uniprot database, and the sequence was imported into Expasy (https://web.expasy.org/ ) website to calculate the isoelectric point of the protein (20 (link)). Then, the preconcentration experiment was carried out to determine the optimal coupling conditions. The target protein was coupled to CM5 chip (GE Healthcare) using the Immobilization module in Biacore T200 Control Software, and the reference channel was set to deduct the background. The chip was activated by EDC/NHS (GE Healthcare), and the uncoupling site was blocked by ethanolamine (GE Healthcare).
sitagliptin (HPLC ≥ 98%, Shanghai yuanye Bio-Technology Co., Ltd) was dissolved in DMSO (VWR) solution and diluted with PBS-P+ (GE Healthcare) solution to the desired compound concentration (1×PBS-P+, 5% DMSO). The affinity and kinetics of sitagliptin with the novel target protein were tested by LMW kinetics module in Biacore T200 Control Software, and extra wash after injection with 50% DMSO was added.
Firstly, the amino acid sequence of the target protein was searched by Uniprot database, and the sequence was imported into Expasy (
sitagliptin (HPLC ≥ 98%, Shanghai yuanye Bio-Technology Co., Ltd) was dissolved in DMSO (VWR) solution and diluted with PBS-P+ (GE Healthcare) solution to the desired compound concentration (1×PBS-P+, 5% DMSO). The affinity and kinetics of sitagliptin with the novel target protein were tested by LMW kinetics module in Biacore T200 Control Software, and extra wash after injection with 50% DMSO was added.
5
SPR-binding Assay for CBP BrD Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SPR-binding assays were performed on a Biacore T200 instrument (GE Healthcare, Chicago, IL, USA) at 25 °C, as previously described [30 (link), 31 (link)]. CBP BrD protein was covalently immobilized on a CM5 chip using a standard amine-coupling procedure in 10 mM sodium acetate (pH 5.0). The chip was first equilibrated in HBS buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 0.05% (v/v) surfactant P20, and 0.2% (v/v) DMSO) overnight. The compounds were serially diluted with HBS buffer and injected for 120 s (contact phase), followed by 240 s (dissociation phase). The KD values of the tested compounds were determined by Biacore T200 evaluation software (GE Healthcare, Chicago, IL, USA).
6
Repebody Binding Affinity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Binding affinity of a repebody was determined through surface plasmon resonance (Biacore T200, GE Healthcare). Briefly, 250 μg/mL of NeutrAvidin Protein (Thermo Scientific, Waltham, USA) was first coated on the surface of CM5 chip (GE Healthcare) in 10 mM sodium acetate buffer (pH 4.5). After immobilization, 20 μg/mL of biotinylated HER2 ectodomain was injected into the chip. Sensorgrams were obtained by flowing a serially diluted repebody into the chip. Kinetic constants were determined by the 1:1 Langmuir binding model using Biacore T200 software (GE Healthcare).
7
Binding Affinities of Ero1, Osm1, and Chimeric Osm1 Analyzed by SPR and ITC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SPR experiments were performed using a BIACORE T-200 (GE Healthcare) equipped with a Series CM5 sensor chip. MBP-Ero1 (83 kDa, > 90% pure based on SDS-PAGE) was immobilized using amine-coupling chemistry as indicated in the BIACORE T-200 wizard program. MBP-Ero1 at a concentration of 30 µg/mL in 10 mM sodium acetate, pH 5.0, was immobilized at a density of 1500RU. A reference cell was prepared by blocking with ethanolamine. To analyze the binding affinity to Ero1, Osm1 or chimeric Osm1 in PBS was injected over the two flow cells at concentrations ranging from 6.25 to 200 µM at a flow rate of 30 µL/min at 25 °C. The BIACORE T-200 evaluation software (GE Healthcare) was used for data processing and the Kd value was calculated by affinity fitting.
For affinity analysis between Osm1 and FAD or Ero1, isothermal titration calorimetry was used. For the interaction between Osm1 and Ero1, 2.5 μL of 1.6 mM Osm1 was injected into a nano-ITC cell (TA Instrument) containing 40 µM Ero1 in PBS. For the interaction between free FAD and Osm1, 2.5μL of 1 mM FAD was injected into nano-ITC cell containing 30 µM holo-Osm1 or 28 µM S78/P162R double mutant.
For affinity analysis between Osm1 and FAD or Ero1, isothermal titration calorimetry was used. For the interaction between Osm1 and Ero1, 2.5 μL of 1.6 mM Osm1 was injected into a nano-ITC cell (TA Instrument) containing 40 µM Ero1 in PBS. For the interaction between free FAD and Osm1, 2.5μL of 1 mM FAD was injected into nano-ITC cell containing 30 µM holo-Osm1 or 28 µM S78/P162R double mutant.
8
SPR Assay of Nur77-LBD and B6
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following the same procedure in our previous study (Ao et al., 2022 (link)). A BIAcore T200 instrument (GE Healthcare) was used in the SPR study. The binding kinetics of Nur77-LBD and B6 were analyzed using the BIAcore T200 (GE Healthcare) at 25°C. The screening concentration was from 0.28 to 10 μM. The negative control was phosphate-buffered saline (PBS). Nur77-LBD proteins were diluted to 0.4 mg/mL in NaOAc (pH 4.5) and immobilized using amine coupling at 6,000 receptor units (RU) on a CM5 sensor chip (GE Healthcare). B6 was injected into the flow wells in running buffer (PBS, 0.1% DMSO) at a flow rate of 30 mL/min for 120 s of association, then dissociated for 420 s. The data was analyzed using the BIAcore T200 Evaluation Software 2.0. The dissociation constant (KD) was calculated using kinetic data from gradient concentrations fitted to a 1:1 interaction model.
9
Tf-TfR Binding Interaction Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tf-TfR interactions in the presence or absence of the test samples were analyzed based on surface plasmon resonance using Biacore T200 (GE Healthcare, Buckinghamshire, UK). The test samples analyzed were pabinafusp alfa, anti-hTfR mAb, and anti-hTfR polyclonal antibody (anti-hTfR pAb; R&D Systems, Minneapolis, MN). The samples were diluted with running buffer (HBS-P+; GE Healthcare) to a final concentration of 100 nmol/L (capture solutions). The extracellular domain of hTfR (a recombinant protein produced in-house using Chinese hamster ovary cells) was bound to each test sample for saturation prior to the addition of human holo-Tf (R&D Systems). The final concentration of hTf was 200 nmol/L. A Series S Sensor Chip NTA (GE Healthcare) was docked on the Biacore T200 and equilibrated with the running buffer, and measurements were made under standard conditions. The relative binding ratio was quantified by calculating the ratio of the Tf-TfR binding response (resonance unit) in the presence of test samples to the Tf-TfR binding response without test samples but with human IgG1 (Sigma-Aldrich, St. Louis, MO).
10
Surface Plasmon Resonance of PTGIS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All SPR measurements were performed at 25 °C in a four-channel optical biosensor Biacore T-200 (GE Healthcare, USA) operated using the Biacore T-200 Control Software. The biotinylated PTGIS was noncovalently immobilized on the streptavidin-coated chip (SA) in the working channel up to 4000 RU (about 4 ng of a bound protein). Buffer A was used as a running buffer. It should be pointed out that PTGIS preparation was immobilized mainly as monomers because in additional experiments we did not observe the significant biosensor signal decrease under harsh regeneration conditions.
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!