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Proteon xpr36

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The ProteOn XPR36 is a label-free, real-time biomolecular interaction analysis system developed by Bio-Rad. The system is designed to simultaneously monitor multiple biomolecular interactions in real-time, providing insights into binding kinetics and affinities.

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Lab products found in correlation

Proteon manager software, by Bio-Rad (21 mentions) Glc sensor chip, by Bio-Rad (8 mentions) Glc chip, by Bio-Rad (6 mentions) Nunc immuno plate, by Thermo Fisher Scientific (6 mentions) Tween 20, by Merck Group (4 mentions) Neutravidin nlc sensor chip, by Bio-Rad (2 mentions) Glh sensor chip, by Bio-Rad (2 mentions) Sodium acetate buffer, by Bio-Rad (2 mentions) Prism v6.0h, by GraphPad (2 mentions) Biaevaluation software, by GE Healthcare (2 mentions) Hbs ep ph 7.4 running, by Teknova (2 mentions) Biacore t200, by GE Healthcare (2 mentions) Ez link maleimide peg2 biotin, by Thermo Fisher Scientific (2 mentions) Neutravidin sensor chip, by Bio-Rad (2 mentions) Biacore 8k, by Bio-Rad (2 mentions) Human c3b, by Complement Technology (2 mentions) Biacore 8k, by GE Healthcare (2 mentions) Proteon manager version 2, by Bio-Rad (2 mentions) Proteon nlc sensor chips neutravidin coated sensor chip, by Bio-Rad (2 mentions) Proteon software package, by Bio-Rad (2 mentions)

106 protocols using proteon xpr36

1

Surface Plasmon Resonance Analysis of C5 Binding

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Surface plasmon resonance (SPR) assay was performed on a Bio-Rad ProteOn XPR36 (Bio-Rad, Hercules, CA, USA) or a Biacore 8K (formerly GE Healthcare, now part of Cytiva, Marlborough, MA, USA). Human complement C5 proteins (wt or R885 variants) were immobilized on a Bio-Rad GLH sensor chip docked in ProteOn or on a CM5 sensor chip docked in Biacore 8K, followed by flowing of zilucoplan or eculizumab biosimilar at varying concentrations in 1× HEPES buffer (pH 7.4, 150 mM of NaCl, 1 mM of MgCl2, 0.005% surfactant P-20, and 1% dimethyl sulfoxide (DMSO) or 1× phosphate-buffered saline (PBS) buffer (pH 7.4, 0.005% P-20, and 1% DMSO). The resulting SPR sensorgrams were recorded and analyzed using the software provided by the vendors to extract the association and dissociation rate constants (ka and kd) and the binding affinity (KD).
For the binding of C5 and C3b, human C3b (Complement Technology) was site-specifically biotinylated via the thioester using Thermo Fisher EZ-link™ maleimide-PEG2-biotin. Biotinylated C3b was immobilized on a Bio-Rad neutravidin sensor chip. C5 in the absence and presence of zilucoplan in 1×HEPES buffer pH 7.4 was flowed over the immobilized C3b, and the resulting SPR signals were recorded using a Bio-Rad ProteOn XPR36.
Tang G.Q., Tang Y., Dhamnaskar K., Hoarty M.D., Vyasamneni R., Vadysirisack D.D., Ma Z., Zhu N., Wang J.G., Bu C., Cong B., Palmer E., Duda P.W., Sayegh C, & Ricardo A. (2023). Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation. Frontiers in Immunology, 14, 1213920.
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2

Surface Plasmon Resonance Analysis of C5 Binding

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Surface plasmon resonance (SPR) assay was performed on a Bio-Rad ProteOn XPR36 (Bio-Rad, Hercules, CA, USA) or a Biacore 8K (formerly GE Healthcare, now part of Cytiva, Marlborough, MA, USA). Human complement C5 proteins (wt or R885 variants) were immobilized on a Bio-Rad GLH sensor chip docked in ProteOn or on a CM5 sensor chip docked in Biacore 8K, followed by flowing of zilucoplan or eculizumab biosimilar at varying concentrations in 1× HEPES buffer (pH 7.4, 150 mM of NaCl, 1 mM of MgCl2, 0.005% surfactant P-20, and 1% dimethyl sulfoxide (DMSO) or 1× phosphate-buffered saline (PBS) buffer (pH 7.4, 0.005% P-20, and 1% DMSO). The resulting SPR sensorgrams were recorded and analyzed using the software provided by the vendors to extract the association and dissociation rate constants (ka and kd) and the binding affinity (KD).
For the binding of C5 and C3b, human C3b (Complement Technology) was site-specifically biotinylated via the thioester using Thermo Fisher EZ-link™ maleimide-PEG2-biotin. Biotinylated C3b was immobilized on a Bio-Rad neutravidin sensor chip. C5 in the absence and presence of zilucoplan in 1×HEPES buffer pH 7.4 was flowed over the immobilized C3b, and the resulting SPR signals were recorded using a Bio-Rad ProteOn XPR36.
Tang G.Q., Tang Y., Dhamnaskar K., Hoarty M.D., Vyasamneni R., Vadysirisack D.D., Ma Z., Zhu N., Wang J.G., Bu C., Cong B., Palmer E., Duda P.W., Sayegh C, & Ricardo A. (2023). Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation. Frontiers in Immunology, 14, 1213920.
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3

Characterization of eboIZN39IQ Native Conformation

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Example 2

To validate that eboIZN39IQ presents the native conformation of the N-trimer found in the prehairpin intermediate, binding to its native ligand, the ebolavirus C-peptide, was characterized (FIG. 2), which binds along the entire groove of the N-trimer in the post-fusion trimer-of-hairpins conformation. Surface plasmon resonance (SPR) analysis (ProteOn XPR36, Bio-Rad) of the interaction of eboC37 with eboIZN39IQ showed a dissociation constant of 14 nM (FIG. 4), with no binding to the D3 negative control. This tight binding affinity is of the same magnitude as the HIV-1 N-trimer/C-peptide interaction(28) and indicate that eboIZN39IQ presents a native N-trimer. A shortened C-peptide (eboC24), missing the 13 N-terminal residues of eboC37, bound to eboIZN39IQ with a dissociation constant of ˜300 nM and did not bind to the D3 negative control (FIG. 11).

US10189878B2. Ebolavirus pre-hairpin intermediate mimics and methods of use (2019-01-29). UNIVERSITY OF UTAH RESEARCH FOUNDATION [US], None [US]. Inventors: Tracy R. Clinton [US], Michael Thomas Jacobsen [US], Matthew T. Weinstock [US], Brett D. Welch [US], Debra Muir Eckert [US], Michael S. Kay [US].
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4

Aptamer-mediated Protein Binding Kinetics

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For binding affinity measurements, the surface plasmon resonance (SPR) technology-based ProteON XPR36 (Bio-Rad, Hercules, CA) was used with PBS (pH 7.4) at 25°C. Biotinylated ssDNA aptamers (0.25 μg/μL) in PBS were immobilized on a NeutrAvidin (NLC) sensor chip (Bio-Rad) in vertical orientation at a flow rate of 30 μL/min.
Concentrations of 0.625, 1.25, 2.5, 5, and 10 μM H1-HA1 protein in PBS were run across the surface in horizontal orientation at a flow rate of 100 μL/min for 60 s with a dissociation time of 600 s. Data were analyzed with the ProteON Manager software, and binding constants were determined using a simple 1:1 Langmuir model. Equilibrium dissociation constants (KD) were calculated from association and dissociation rate constants (KD = kd/ka).
Woo H.M., Lee J.M., Yim S, & Jeong Y.J. (2015). Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5. PLoS ONE, 10(4), e0125060.
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5

Investigating Chaperone Protein Interactions

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The interaction between PfHsp70-1 with its functional partners, PfHsp70-z and PfHop, was investigated using a Bio-Rad ProteOn XPR36 surface plasmon resonance (SPR) system (Hercules, CA, USA), as previously described [16 (link),30 (link)]. Protein immobilisation was conducted on a GLC chip. As ligands, PfHsp70-1 and its subdomain, PfHsp70-1NBD, were immobilised on the chip to achieve 178 and 188 response units, 190 response units for PfHsp70-z, and 197 response units for PfHop, respectively. As analytes, aliquots of PfHsp70-1, PfHsp70-1NBD, PfHsp70-z, and PfHop were prepared at 0, 125, 250, 500, 1000 and 2000 nM and injected at 50 μL/min on each horizontal surface. Data collected was double referenced using a buffer blank (buffer without protein) and a channel in which BSA was immobilised as a non-chaperone protein control. The assay was repeated in the presence of nucleotides (either 5 mM ADP or 5 mM ATP) as controls [16 (link),30 (link)]. To determine the effects of EGCG on the protein-protein interaction, the assays were repeated in the presence of 2.5 μM EGCG. The SPR data were analysed as previously described [16 (link),30 (link)].
Zininga T., Ramatsui L., Makhado P.B., Makumire S., Achilinou I., Hoppe H., Dirr H, & Shonhai A. (2017). (−)-Epigallocatechin-3-Gallate Inhibits the Chaperone Activity of Plasmodium falciparum Hsp70 Chaperones and Abrogates Their Association with Functional Partners. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(12), 2139.
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6

SPR Binding Kinetics of Annexin A2

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SPR studies were performed using a BioRad ProteOn XPR36 instrument according to a One-shot Kinetics protocol [51 (link), 52 (link)]. GLC chips were initialized using glycerol and preconditioning was performed by sequential injections of 0.5% SDS, 100 Mm HCl, and then 50 mM NaOH, each for 60 sec at 25 μl/min. The surface was then activated using 40 mM EDAC and 10 mM Sulfo-NHS, injected for 5 min at 25 μl/min. 50 μg/ml of Annexin A2 in 10 mM sodium acetate, pH 5.5, was injected in the vertical direction over the desired channels for 6 min at 25 μl/min. 50 μg/ml of BSA was also immobilized in the same way using as a control. The remaining activated carboxyl groups were blocked with a 3 min injection of 1 M ethanolamine at 25 μl/min in the vertical direction. The chip was washed with PBS until a stable baseline was achieved. The running buffer was then switched to TBS with 0.05‰ Tween-20 (TBS-T). A concentration series of the analyte of interest was injected over the immobilized ligand (Annexin A2). Injection of the analyte diluted in TBS-T was done in the horizontal direction at 50 μl/min for 3 min. An equivalent buffer injection was used for reference subtraction. The dissociation time was set to 12 min. Equilibrium and rate constants were calculated using ProteOn Manager Software. A local Rmax value and the Langmuir model were used.
Cui H.Y., Wang S.J., Miao J.Y., Fu Z.G., Feng F., Wu J., Yang X.M., Chen Z.N, & Jiang J.L. (2015). CD147 regulates cancer migration via direct interaction with Annexin A2 and DOCK3-β-catenin-WAVE2 signaling. Oncotarget, 7(5), 5613-5629.
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7

Kinetic Analysis of sdAb-DENV NS1 Interactions

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Affinity and kinetics measurements were performed using the ProteOn XPR36 (Bio-Rad). Lanes of a general layer compact (GLC) chip were individually coated with DENV NS1 of each serotype or NS1 of West Nile virus (WNV) or Zika virus (ZIKV). Immobilization of the proteins were performed using dilution to 20 µg/mL in 10 mM acetate buffer pH 5.0 and attached to the chip following the standard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/ N-hydroxysulfosuccinimide (sulfo-NHS) coupling chemistry available from the manufacturer. Binding kinetics of each sdAb was tested at 25 °C by flowing six concentrations typically varying from 300 to 0 nM at 100 μL/min for 90 s over the antigen coated chip and then monitoring dissociation for 600 s. Following each run, the chip was regenerated by flowing 0.085% phosphoric acid (~pH 3.0) across the surface for 18 s. Data analysis was performed with ProteOn Manager 2.1 software, corrected by subtraction of the zero antibody concentration column as well as interspot correction. The standard error on the fits was less than 10%. Binding constants were determined using the Langmuir model built into the analysis software. Similar assays were performed where selected sdAb were immobilized in a like manner to the GLC sensor chip and then tested for their ability to bind dilutions of the various NS1 proteins.
Shriver-Lake L.C., Liu J.L., Zabetakis D., Sugiharto V.A., Lee C.R., Defang G.N., Wu S.J., Anderson G.P, & Goldman E.R. (2018). Selection and Characterization of Anti-Dengue NS1 Single Domain Antibodies. Scientific Reports, 8, 18086.
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8

Kinetic Binding Analysis of PBRM Conjugate

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Example 47

The kinetic binding of the PBRM-polymer-drug conjugate to an immobilized receptor is determined by BIAcore SPR. The binding constants for the PBRM in the PBRM-polymer-drug conjugate and PBRM alone can be determined using standard BIAcore procedures.

Using standard amine coupling chemistry, hErbB2 is immobilized in three flow channels to the surface Plasmon resonance sensor chip surface at three similar densities, trastuzumab readily bound to the immobilized hErbB2 thereby demonstrating that both binding partners were active. The binding parameters ka (association or affinity rate constant) and KD(dissociation constant) are measured at 25° C. for the PBRM-polymer-drug conjugate and PBRM using a BioRad ProteOn XPR36 optical biosensor equipped with a GLC sensor chip and equilibrated with running buffer.

The results show that the PBRM in the PBRM-polymer-drug conjugate is recognized by the PBRM receptor and that the binding of the PBRM in the PBRM-polymer-drug conjugate is not significantly affected relative to the unconjugated PBRM.

US11434278B2. Protein-polymer-drug conjugates (2022-09-06). Asana BioSciences, LLC [US]. Inventors: Roger A. Smith [US], Nitin K. Damle [US], Aleksandr V. Yurkovetskiy [US], Mao Yin [US], Timothy B. Lowinger [US], Joshua D. Thomas [US], Cheri A. Stevenson [US], Venu R. Gurijala [US].
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9

Nanobody Binding to CTLA-4 in Primates

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Example 1

Monovalent F023700906 Nanobody 11F01 (L11V,A14P,Q45R,A74S,K83R, V89L,M96P,Q108L)-FLAG3-HIS6), a building block of F023700912, demonstrates binding to CTLA-4-Fc fusion molecule from both human and cynomolgus monkey. On-rate, off-rate and affinity were determined on a ProteOn XPR36 (BioRad 670BR0166) using human CTLA-4-hFc and cynomolgus monkey CTLA4-hFc (Table D below). These results demonstrate high-affinity binding of the Nanobody to human and cynomolgus monkey CTLA-4 suggesting potential for the Nanobody to modulate the function of CTLA-4 and that cynomolgus monkey may be used as a toxicology species.

TABLE D-1
Nanobody Binding to Human or Cynomolgous Monkey CTLA-4-Fc
Human CTLA-4-FcCynomolgus CTLA-4-Fc
Ka (1/Ms)Kd (1/s)KD (M)Ka (1/Ms)Kd (1/s)KD (M)
F0237009064.8E+065.9E−031.2E−094.7E+065.7E−031.2E−09

TABLE D-2
Reagents
ExpressionConc.
Reagentsystem(mg/ml)Formulation BufferSEC purityRP purity
hCTLA4-FcHEK293F16.1310 mM Sodium94.86%61.50%
Phosphate, 75 mM(~150 KDa
NaCl, 3% Sucrose,(tetramer)
pH = 7.4
cynoCTLA4-HEK293EBNA0.32PBS pH 7.481.58%83.50%
Fc

US10501542B2. CTLA4 binders (2019-12-10). Merck Sharp & Dohme Corp. [US]. Inventors: Juha Punnonen [US], Maribel Beaumont [US], Marie-Ange Buyse [BE], Carlo Boutton [BE], Bruno Dombrecht [BE], Bjorn Victor [BE], Robert A. Kastelein [US].
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10

PAR4 Antibody Binding Specificity

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Example 3

In order to determine the specificity of the anti-hPAR4 clones to PAR4, ELISA screening was performed as described above. FIG. 5A shows the result for binding specificity of clones mAb-5ARC3 (5A.RC3) and mAb-5BRB4 (5B.RB4) against hPAR1, hPAR2, hPAR3 and hPAR4.

5A.RC3 demonstrated a 16-fold selectivity to human PAR4 peptide over PAR1, PAR2 and PAR3. mAb 5B.RB4 bound to all four human PAR peptides similarly and with lower affinity to PAR4 compared to 5A.RC3.

The inventors performed surface plasmon resonance (SPR) analysis utilising the Bio-Rad Proteon XPR36 to gain insight into the binding affinity (rate of association and dissociation) and specificity of 5A.RC3.F10b.H4b. Utilising a streptavidin chip, all biotin-coupled human PAR peptides (Table 1) were captured on the surface and different concentrations of purified 5A.RC3 passed over. Clone 5A.RC3.F10b.H4b was observed to have a dissociation constant (KD) of about 0.4 nM by SA chip SPR (FIG. 5B).

US11780915B2. Binding proteins to the human thrombin receptor, PAR4 (2023-10-10). Monash University [AU]. Inventors: Justin Hamilton [AU], Mark Sleeman [AU].
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