Protein interaction was monitored in real-time by interferometry Bio-Layer detection with the BLItz System from Pall forte BIO, USA. Recombinant LRP51407–1615-HA-(His)6 was immobilized on a Ni-NTA sensor (#18-5101) and excess protein was removed by washing with PBS. For the Kon determination, TRAF6-Flag (Origene, USA #:TP319528) was either added at a concentration of 167 nM or 822 nM, and Koff was determined by washing with PBS until a stable binding curve was achieved.
Blitz system
Manufactured by Molecular Devices
Sourced in United States, Spain
The BLItz system is a label-free, real-time molecular interaction analysis instrument that measures binding kinetics and affinity. It utilizes biolayer interferometry (BLI) technology to monitor biomolecular interactions in real-time without the need for labeling. The BLItz system provides a compact and versatile solution for a wide range of applications, including protein-protein, protein-small molecule, and protein-nucleic acid interactions.
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Lab products found in correlation
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Ni nta biosensor, by Molecular Devices (10 mentions)
Blitz system software, by Molecular Devices (7 mentions)
Blitz pro 1, by Molecular Devices (4 mentions)
B9806, by Merck Group (3 mentions)
His tag biosensors, by Molecular Devices (3 mentions)
High precision streptavidin biosensors, by Molecular Devices (2 mentions)
Ce501, by Merck Group (2 mentions)
B1557, by Merck Group (2 mentions)
Dip and read streptavidin, by Molecular Devices (2 mentions)
Dip and read anti human igg fc capture, by Molecular Devices (2 mentions)
Dip and read anti higg fc, by Molecular Devices (2 mentions)
Prism program, by GraphPad (2 mentions)
Recombinant human fc fusion ace2, by Sino Biological (2 mentions)
Blitz pro software, by Molecular Devices (2 mentions)
Blitz pro, by Molecular Devices (2 mentions)
Streptavidin coated biosensors, by Molecular Devices (2 mentions)
Blitz software, by Molecular Devices (2 mentions)
Streptavidin sa biosensor, by Molecular Devices (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
112 protocols using blitz system
1
Real-time LRP5-TRAF6 Interaction Kinetics
2
Kinetic Analysis of Transcriptional Regulators
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The kinetic parameters of the interaction, binding affinity (KD), and rate constants of association (ka) and dissociation (kd) between PsdR or ApsR and DNA fragments encompassing predicted promoter binding sites (PpsdA, PderA, Pdlt and PmprF; see Supplementary Table S3 ) or a non-specific DNA fragment used as a negative control (Flta), were measured by BLI using the BLITz system (FortéBio). To this purpose, 100 bp DNA fragments were synthesized by PCR using biotinylated reverse primers (Supplementary Table S2 ; Isogen Life Science), purified and dissolved in distilled water. The concentrations of the DNA fragments were measured by using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and adjusted to 40 ng ml−1. Streptavidin biosensors (ForteBio) were equilibrated in BB for 30 min followed by the binding of the corresponding DNA fragment to the biosensor until saturation (15 min). Before the assays, His-tagged ApsR or PsdR were incubated in BB buffer supplemented with acetyl-phosphate (50 mM) for ApsR or ammonium phosphoramidate (50 mM) for PsdR, and MgCl2 (100 mM) for 30 min and conveniently diluted with BB. For each interaction, five dilutions of the protein plus a reference without protein added were assayed. Kinetics values calculation and data analysis were performed with BLItz Pro 1.2 software using a 1:1 stoichiometry model.
3
Binding Kinetics of CCHFV N Protein
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Biolayer interferometry was used to monitor the binding affinities of wild type CCHFV N protein and its stalk domain with the 5’ untranslated regions (5’ UTR) of CCHFV S-segment derived mRNA, using the BLItz system (ForteBio Inc.), as previously reported [22 (link)–24 (link)]. Briefly, the biotin- 5’ UTR was synthesized and loaded onto high precision streptavidin biosensors (catalog # 18–5019, Forte Bio Inc.), as previously reported [20 (link)]. All reactions were carried out at room temperature in RNA binding buffer (20 mM Tris-HCl, pH 7.4, 80 mM NaCl, 20mM KCl, and 1mM DTT). After mounting the RNA, the biosensors were equilibrated in RNA binding buffer and then dipped in the purified protein solutions of either wild type N protein or stalk domain for the measurement of association kinetics. The reaction cycles were as follows: initial base line for 30 seconds, loading of biotinylated RNA on streptavdin biosensors for 120 seconds, base line for 30 seconds, association of protein with the RNA for 300s, followed by dissociation phase of 500 seconds. The kinetic parameters Kass (association rate constant), Kdis (dissociation rate constant) and the binding affinities (Kd = Kdis/Kass) were calculated with the help of inbuilt data analysis software (BLItZ Pro), as previously reported [22 (link)–24 (link)].
4
Heparin Binding Affinity of TcdA
5
Biolayer Interferometry of Protein-Lipid Interactions
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Protein–lipid interactions were determined using the biolayer interferometry from the BLItz system (Pall ForteBio, USA). Protein–lipid interactions were observed by immobilizing 500 µM of biotinylated liposomes on a streptavidin biosensor. After immobilization, the sensor was washed with buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl, and 0.1% BSA to prevent nonspecific association. In total, 25 µM of proteins were added to the sensor and the change in binding (nm) was measured. Proteins were then allowed to disassociate from the probe in the buffer previously mentioned. The data were processed and plotted using the Prism Software.
6
Binding and Signaling of MPS IIIA GAGs and FGF2
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The binding of MPS IIIA GAGs to FGF2 was analysed in the Blitz ® system (ForteBio) using protein G sensors and operated with PBS, pH 7.4 containing 1% (v/v) Tween-20. Sensors were equilibrated in buffer for 30 s and then exposed to anti-FGF2 antibody (1:1000, rabbit polyclonal anti-FGF2, R&D Systems) for 120 s followed by rinsing in buffer for 30 s. Sensors were exposed to 50 μg/mL FGF2 for 120 s followed by rinsing in buffer for 30 s and then exposed to MPS IIIA GAGs or heparin (1–20 μg/mL; Sigma H3393) for 120 s and rinsed with buffer for 30 s. The affinity, KD, between FGF2 and either MPS IIIA GAGs or heparin was determined assuming 1:1 binding. Signalling via the fibroblast growth factor receptor (FGFR) was determined using the BaF32 cell proliferation assay which relies on the formation of an active ternary complex between GAG, FGF2 and FGFR1c [44] (link). The assay was performed as described previously [45] (link) in the presence of 0.03 nM FGF2 and either unfractionated heparin (0–0.5 μg/mL; Sigma H3393) or MPS IIIA GAG (0–2 μg/mL). The relative number of cells present was determined using the MTS reagent (Promega, Madison, Wisconsin, USA) by addition to the cell cultures for 6 h prior to measurement of absorbance at 490 nm.
7
Quantifying FMRP-RNA Binding Kinetics
8
Kinetics of Dut-Stl Interaction by BLI
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The kinetics parameters of the interaction, binding affinity (KD) and rate constants of association (kon) and dissociation (koff), between Duts and Stl were measured by biolayer interferometry (BLI) using the BLITz system (FortéBio). Proteins were diluted in buffer STL and the assays were carried out in the same buffer, when necessary it was supplemented with the corresponding uracil nucleotide, 0.5 mM of dUPNPP or 5 mM or dUMP. A non-reducing buffer was used to evaluate the interaction of Dut80αD81A C-C with Stl (400 mM NaCl, 75 mM HEPES pH7.5, 5 mM MgCl2). Biosensor hydration, baselines and dissociation analysis were carried out in buffer STL without nucleotide addition. For each interaction, the corresponding His-tagged Dut was immobilized on Ni-NTA biosensors (FortéBio) at 1 μM concentration. At least five different dilutions of Stl (from 4 to 0.062 μM plus the reference without Stl) were used in the association and dissociation steps for each Stl:Dut interaction measured, adjusting the highest concentration of Stl to 10 times the estimated KD (Table 1 ). Kinetics values calculation and data analysis were performed with BLItz Pro 1.2 software. A 1:1 model was employed to fit the data.
9
Quantifying TcsL-SEMA Protein Interactions
10
Measuring TcdB-FZD CRD Binding Affinities
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The binding affinities between TcdB and FZD-CRDs were measured by BLI assay using the Blitz system (ForteBio). Briefly, the CRDs-Fc of FZD1, 2, 5, 7 or human IgG1 Fc (20 μg/ml) were immobilized onto capture biosensors (Dip and Read Anti-hIgG-Fc, ForteBio) and balanced with PBS. The biosensors were then exposed to TcdB or TcdB1-1830, followed by washing with PBS. Binding affinities (KD) were calculated using the Blitz system software (ForteBio).
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