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  • 87
    Enamine Ltd spr measurement
    Spr Measurement, supplied by Enamine Ltd, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/spr measurement/product/Enamine Ltd
    Average 87 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    86
    Millipore spr measurements
    Mismatch binding and sliding clamp formation by dimeric MutS. ( A ) Quantitative measurements of binding of dimeric MutS D835R to 21-bp <t>DNA</t> of different sequences (see Supplementary Table S1 ) as determined with <t>SPR.</t> Measured values for k off [determined with Evilfit ( 38 , 39 )] are plotted against [determined with Graphpad Prism ( 37 ) as specified in the ‘Materials and Methods’ section] in the absence (left graph) and the presence (right graph) of ATP for different mismatches. Values for k off larger than 0.4 s −1 could not be determined and were plotted on the dotted line at 0.4 s −1 . Red triangles indicate measurements for binding homoduplex sequences. ( B ) Variation of flanking DNA sequences around a GT mismatch is schematically represented by colors. To achieve blocked DNA ends, anti-fluorescein antibodies were bound to fluorescein moieties (both represented in red) that were coupled to the DNA strands. ssDNA linkers attaching the DNA duplex to the surface of the chip are indicated by dashed lines. Measured values for k off are plotted against in the absence (left graph) and the presence (right graph) of ATP for binding to a GT mismatch in the context of different flanking sequences. ( C ) MutS sliding clamp formation was investigated for three mismatches by binding dimeric MutS D835R to unblocked DNA, and subsequently releasing the MutS with buffer with increasing ATP concentrations at the time point indicated by the red arrow (top three graphs). Using fixed kinetics for the slow release observed in absence of ATP, the contribution of a faster release corresponding to the percentage of sliding clamps formed could be estimated for each ATP concentration (bottom graph).
    Spr Measurements, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/spr measurements/product/Millipore
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    86
    Biacore spr measurement analysis
    Determination of binding constant, kinetics and thermodynamic parameters of the <t>LigBCen2R/GBD</t> interaction by ELISA, <t>SPR,</t> and ITC. (A) Binding of serial concentrations of LigBCen2NR to immobilized GBD by ELISA. Serial concentrations of GST-LigBCen2R, GST-LigBCen2NR, GST-LigBCen2 (positive control), or GST-LigCon (negative control) were added to 1 µM of GBD or BSA coated wells (negative control, data not shown). (B) SPR analysis of LigBCen2R interacting with GBD. 1.5 µM of Recombinant Histidine-tagged LigBCen2R was immobilized on the surface of a Ni-NTA chip. GBD in Tris Buffer containing 100 µM CaCl2 at pH 7.5 flowed through the chip and the concentrations of GBD ranged from 40 to 0.625 µM (from top to bottom). The KD, kon, and koff were obtained from the average of duplicate experiments shown in Table 1 . (C) Determination of the binding affinity by ITC. The cell contained 1 ml of GBD and the syringe contained 250 µl of LigBCen2R (upper panel). Heat differences obtained from 25 injections of LigBCen2R; (lower panel). Integrated curve with experimental data (⋄) and the best fit (-). The thermodynamic parameters are shown as the average of duplicate experiments (KD = 1.88±0.09 µM, ΔH = −29.25±3.58 kcal mol-1, TΔS = −21.47±3.61 kcal mol-1 K-1, n = 0.995±0.01). (D) Fluorescence spectrum of Alexa-488 labeled LigBCen2RW1073C in the presence and absence of GBD. One µM of Alexa-488 labeled LigBCen2RW1073C in Tris buffer was excited at 485 nm. Aliquots of GBD from respective stock solutions were added. The figure shows Alexa488 fluorescence in the presence of 0, 1.62, 3.12, 6.25, 12.5, 25 µM of GBD (Inner plot). The determination of KD of Alexa488 labeled LigBCen2RW1073C and GBD by monitoring the quenching fluorescence intensity of Alexa488 labeled LigBCen2RW1073C titrated by GBD. The emission wavelength recorded in this figure was 513 nm, and KD was revealed by fitting the data point into the equation described in materials and methods (KD = 1.93.4 µM).
    Spr Measurement Analysis, supplied by Biacore, used in various techniques. Bioz Stars score: 86/100, based on 215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biacore biacore spr measurement
    <t>BIAcore</t> quantification of p55 interaction with peptides encoded by exons 5 and 10. The Surface Plasmon Resonance <t>(SPR)</t> technique was used to quantify the interaction of p55 with the FERM domain of protein 4.1R with and without exon 5-peptide (A–C). The same technique was used to demonstrate the non-competition of exon 5 and exon 10 peptide-binding sites to His-p55 (D). Sensograms were obtained from SPR analysis of the interaction between His-p55 and the FERM domain constructs of protein 4.1R. Recombinant His-p55 protein was immobilized on the CM5 sensor chip and the MBP-FERM domain fusion proteins were injected as an analyte. The wild type His-p55 protein was expressed either in the bacterial cytoplasm or in Sf9 cells with no effect on its binding properties. (A) The binding properties of MBP-FERM and MBP-FERMΔExon 5 fusion proteins to the immobilized His-p55 were measured at 100 nM concentration of each analyte protein. Sensogram of 100 nM MBP (analyte) binding to His-p55 on the chip was used as a negative control. The sensorgrams were generated using a 30 µl/min analyte flow rate (BIAcore 1000 instrument) and included a 3-minute association and a 5-minute dissociation section. (B) Sensograms of the MBP-FERM domain fusion protein (analyte) injected at various concentrations ranging from 50–400 nM. MBP alone at 500 nM was used as a negative control. The flow rate was 30 µl/min over the immobilized His-p55 surface yielding a k D value of 70 nM. (C) Sensograms of the MBP-FERMΔExon 5 domain fusion protein (analyte) injected at various concentrations ranging from 50–400 nM. The MBP at 500 nM was used as a negative control. The flow rate was 30 µl/min over the immobilized His-p55 surface yielding a k D value of 132 nM. (D) The MBP-exon 5 fusion protein (1 µM) was injected over the His-p55 surface, followed by the washing of the chip surface with the running buffer for 25 min at 5 µl/min flow rate. This step was repeated three times, followed by the injection of 1.0 µM MBP-exon 10 for 25 minutes and washing of the chip surface with the running buffer for 25 minutes. The sensograms show that the exon 10-peptide can bind to the p55 surface that is already saturated with the exon 5-peptide.
    Biacore Spr Measurement, supplied by Biacore, used in various techniques. Bioz Stars score: 92/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Biacore biacore3000 biacore spr measurement
    <t>BIAcore</t> quantification of p55 interaction with peptides encoded by exons 5 and 10. The Surface Plasmon Resonance <t>(SPR)</t> technique was used to quantify the interaction of p55 with the FERM domain of protein 4.1R with and without exon 5-peptide (A–C). The same technique was used to demonstrate the non-competition of exon 5 and exon 10 peptide-binding sites to His-p55 (D). Sensograms were obtained from SPR analysis of the interaction between His-p55 and the FERM domain constructs of protein 4.1R. Recombinant His-p55 protein was immobilized on the CM5 sensor chip and the MBP-FERM domain fusion proteins were injected as an analyte. The wild type His-p55 protein was expressed either in the bacterial cytoplasm or in Sf9 cells with no effect on its binding properties. (A) The binding properties of MBP-FERM and MBP-FERMΔExon 5 fusion proteins to the immobilized His-p55 were measured at 100 nM concentration of each analyte protein. Sensogram of 100 nM MBP (analyte) binding to His-p55 on the chip was used as a negative control. The sensorgrams were generated using a 30 µl/min analyte flow rate (BIAcore 1000 instrument) and included a 3-minute association and a 5-minute dissociation section. (B) Sensograms of the MBP-FERM domain fusion protein (analyte) injected at various concentrations ranging from 50–400 nM. MBP alone at 500 nM was used as a negative control. The flow rate was 30 µl/min over the immobilized His-p55 surface yielding a k D value of 70 nM. (C) Sensograms of the MBP-FERMΔExon 5 domain fusion protein (analyte) injected at various concentrations ranging from 50–400 nM. The MBP at 500 nM was used as a negative control. The flow rate was 30 µl/min over the immobilized His-p55 surface yielding a k D value of 132 nM. (D) The MBP-exon 5 fusion protein (1 µM) was injected over the His-p55 surface, followed by the washing of the chip surface with the running buffer for 25 min at 5 µl/min flow rate. This step was repeated three times, followed by the injection of 1.0 µM MBP-exon 10 for 25 minutes and washing of the chip surface with the running buffer for 25 minutes. The sensograms show that the exon 10-peptide can bind to the p55 surface that is already saturated with the exon 5-peptide.
    Biacore3000 Biacore Spr Measurement, supplied by Biacore, used in various techniques. Bioz Stars score: 87/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Biacore surface plasmon resonance spr assay spr measurement
    AL2-PE38KDEL adaptor-toxin fusion protein and <t>SPR</t> measurements of <t>AL2-PE38KDEL/scFv</t> interaction. ( A ) The AL2-PE38KDEL adaptor-toxin fusion protein has one additional AL fragment linked by a 5-residue linker (Linker2) to the N-terminus of the AL1-PE38KDEL as shown in Fig. 2A . ( B ) SPR measurements of immobilized AL2-PE38KDEL interacting with soluble scFv. The description is the same as in Fig. 2B . The overlaying black lines are the global fit to the experimental data with K d = 5.52 ± 0.03 × 10 −9 M; k on = 3.60 ± 0.06 × 10 5 M −1 S −1 and k off = 1.99 ± 0.04 × 10 −3 S −1 .
    Surface Plasmon Resonance Spr Assay Spr Measurement, supplied by Biacore, used in various techniques. Bioz Stars score: 94/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 142 article reviews
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    84
    Branson Ultrasonics spr
    AL2-PE38KDEL adaptor-toxin fusion protein and <t>SPR</t> measurements of <t>AL2-PE38KDEL/scFv</t> interaction. ( A ) The AL2-PE38KDEL adaptor-toxin fusion protein has one additional AL fragment linked by a 5-residue linker (Linker2) to the N-terminus of the AL1-PE38KDEL as shown in Fig. 2A . ( B ) SPR measurements of immobilized AL2-PE38KDEL interacting with soluble scFv. The description is the same as in Fig. 2B . The overlaying black lines are the global fit to the experimental data with K d = 5.52 ± 0.03 × 10 −9 M; k on = 3.60 ± 0.06 × 10 5 M −1 S −1 and k off = 1.99 ± 0.04 × 10 −3 S −1 .
    Spr, supplied by Branson Ultrasonics, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/spr/product/Branson Ultrasonics
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    spr - by Bioz Stars, 2020-04
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    94
    GE Healthcare spr
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Spr, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/spr/product/GE Healthcare
    Average 94 stars, based on 15 article reviews
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    spr - by Bioz Stars, 2020-04
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    90
    Sensiq Technologies spr
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Spr, supplied by Sensiq Technologies, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/spr/product/Sensiq Technologies
    Average 90 stars, based on 1 article reviews
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    spr - by Bioz Stars, 2020-04
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    93
    GE Healthcare 3000 spr instrument
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    3000 Spr Instrument, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/3000 spr instrument/product/GE Healthcare
    Average 93 stars, based on 62 article reviews
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    90
    GE Healthcare t100 spr instrument
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    T100 Spr Instrument, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t100 spr instrument/product/GE Healthcare
    Average 90 stars, based on 34 article reviews
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    t100 spr instrument - by Bioz Stars, 2020-04
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    93
    Biacore 3000 spr biosensor
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    3000 Spr Biosensor, supplied by Biacore, used in various techniques. Bioz Stars score: 93/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/3000 spr biosensor/product/Biacore
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    91
    Biacore spr sensorgrams
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Spr Sensorgrams, supplied by Biacore, used in various techniques. Bioz Stars score: 91/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millar Inc spr pv catheter
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Spr Pv Catheter, supplied by Millar Inc, used in various techniques. Bioz Stars score: 86/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millar Inc conductance system spr 839
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Conductance System Spr 839, supplied by Millar Inc, used in various techniques. Bioz Stars score: 86/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bionavis bionavis spr
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Bionavis Spr, supplied by Bionavis, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare x spr biosensor system
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    X Spr Biosensor System, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biacore surface plasma resonance spr
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Surface Plasma Resonance Spr, supplied by Biacore, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare 3000 spr system
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    3000 Spr System, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 87/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millar Inc millar spr 524 catheters
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Millar Spr 524 Catheters, supplied by Millar Inc, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biacore t100 spr instrument
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    T100 Spr Instrument, supplied by Biacore, used in various techniques. Bioz Stars score: 92/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Plexera spr imaging instrument
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Spr Imaging Instrument, supplied by Plexera, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biacore 3000a spr instrument
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    3000a Spr Instrument, supplied by Biacore, used in various techniques. Bioz Stars score: 88/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare x100 spr instrument
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    X100 Spr Instrument, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biacore spr device
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Spr Device, supplied by Biacore, used in various techniques. Bioz Stars score: 91/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bionavis mp spr navi 200 instrument
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Mp Spr Navi 200 Instrument, supplied by Bionavis, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare t100 spr system
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    T100 Spr System, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biacore 2000a spr instrument
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    2000a Spr Instrument, supplied by Biacore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biacore 2000 spr instrument
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    2000 Spr Instrument, supplied by Biacore, used in various techniques. Bioz Stars score: 93/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
    Reichert Spr Sr7500 Instrument, supplied by Woojung BSC Inc, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biacore spr real time
    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
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    RAGE binds only to the toxic, prefibrillar form of <t>h-IAPP.</t> ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) <t>SPR</t> shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.
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    Image Search Results


    Mismatch binding and sliding clamp formation by dimeric MutS. ( A ) Quantitative measurements of binding of dimeric MutS D835R to 21-bp DNA of different sequences (see Supplementary Table S1 ) as determined with SPR. Measured values for k off [determined with Evilfit ( 38 , 39 )] are plotted against [determined with Graphpad Prism ( 37 ) as specified in the ‘Materials and Methods’ section] in the absence (left graph) and the presence (right graph) of ATP for different mismatches. Values for k off larger than 0.4 s −1 could not be determined and were plotted on the dotted line at 0.4 s −1 . Red triangles indicate measurements for binding homoduplex sequences. ( B ) Variation of flanking DNA sequences around a GT mismatch is schematically represented by colors. To achieve blocked DNA ends, anti-fluorescein antibodies were bound to fluorescein moieties (both represented in red) that were coupled to the DNA strands. ssDNA linkers attaching the DNA duplex to the surface of the chip are indicated by dashed lines. Measured values for k off are plotted against in the absence (left graph) and the presence (right graph) of ATP for binding to a GT mismatch in the context of different flanking sequences. ( C ) MutS sliding clamp formation was investigated for three mismatches by binding dimeric MutS D835R to unblocked DNA, and subsequently releasing the MutS with buffer with increasing ATP concentrations at the time point indicated by the red arrow (top three graphs). Using fixed kinetics for the slow release observed in absence of ATP, the contribution of a faster release corresponding to the percentage of sliding clamps formed could be estimated for each ATP concentration (bottom graph).

    Journal: Nucleic Acids Research

    Article Title: Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation

    doi: 10.1093/nar/gkt582

    Figure Lengend Snippet: Mismatch binding and sliding clamp formation by dimeric MutS. ( A ) Quantitative measurements of binding of dimeric MutS D835R to 21-bp DNA of different sequences (see Supplementary Table S1 ) as determined with SPR. Measured values for k off [determined with Evilfit ( 38 , 39 )] are plotted against [determined with Graphpad Prism ( 37 ) as specified in the ‘Materials and Methods’ section] in the absence (left graph) and the presence (right graph) of ATP for different mismatches. Values for k off larger than 0.4 s −1 could not be determined and were plotted on the dotted line at 0.4 s −1 . Red triangles indicate measurements for binding homoduplex sequences. ( B ) Variation of flanking DNA sequences around a GT mismatch is schematically represented by colors. To achieve blocked DNA ends, anti-fluorescein antibodies were bound to fluorescein moieties (both represented in red) that were coupled to the DNA strands. ssDNA linkers attaching the DNA duplex to the surface of the chip are indicated by dashed lines. Measured values for k off are plotted against in the absence (left graph) and the presence (right graph) of ATP for binding to a GT mismatch in the context of different flanking sequences. ( C ) MutS sliding clamp formation was investigated for three mismatches by binding dimeric MutS D835R to unblocked DNA, and subsequently releasing the MutS with buffer with increasing ATP concentrations at the time point indicated by the red arrow (top three graphs). Using fixed kinetics for the slow release observed in absence of ATP, the contribution of a faster release corresponding to the percentage of sliding clamps formed could be estimated for each ATP concentration (bottom graph).

    Article Snippet: Unless otherwise indicated, DNA for the SPR measurements (obtained from Sigma) contained a 21-base pair long duplex (see Supplementary Table S1 for the full range of DNA sequences measured for MutS binding) with a single-stranded DNA (ssDNA)-overhang consisting of 20 thymidines ((dT)20 ).

    Techniques: Binding Assay, SPR Assay, Chromatin Immunoprecipitation, Concentration Assay

    DNA binding by the MutS dimer and tetramer. SPR measurements of binding ( A ) wild-type MutS, ( B ) dimeric MutS D835R or ( C ) the cross-linked tetramer of MutS to a G . T mismatch. Different protein concentrations are represented by different colors (legend refers to monomer concentrations) and black lines indicate fitted kinetics using a one-phase binding model (fitted with Biacore T200 Evaluation Software). ( D ) The kinetics of the tetramer of MutS fitted using a model that takes into account bivalent binding and a conformational change, represented in black lines (fitted with Biacore T200 Evaluation Software as specified in the ‘Materials and Methods’ section). ( E ) Determination of for the dimer and tetramer using SPR signal at equilibrium for different protein concentrations binding to the DNA [fitted with Graphpad Prism ( 37 )].

    Journal: Nucleic Acids Research

    Article Title: Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation

    doi: 10.1093/nar/gkt582

    Figure Lengend Snippet: DNA binding by the MutS dimer and tetramer. SPR measurements of binding ( A ) wild-type MutS, ( B ) dimeric MutS D835R or ( C ) the cross-linked tetramer of MutS to a G . T mismatch. Different protein concentrations are represented by different colors (legend refers to monomer concentrations) and black lines indicate fitted kinetics using a one-phase binding model (fitted with Biacore T200 Evaluation Software). ( D ) The kinetics of the tetramer of MutS fitted using a model that takes into account bivalent binding and a conformational change, represented in black lines (fitted with Biacore T200 Evaluation Software as specified in the ‘Materials and Methods’ section). ( E ) Determination of for the dimer and tetramer using SPR signal at equilibrium for different protein concentrations binding to the DNA [fitted with Graphpad Prism ( 37 )].

    Article Snippet: Unless otherwise indicated, DNA for the SPR measurements (obtained from Sigma) contained a 21-base pair long duplex (see Supplementary Table S1 for the full range of DNA sequences measured for MutS binding) with a single-stranded DNA (ssDNA)-overhang consisting of 20 thymidines ((dT)20 ).

    Techniques: Binding Assay, SPR Assay, Software

    States of MutS. ( A ) MutS recognizes a mismatch (pink star) as a dimer. Except when binding homoduplex or a CC mismatch, MutS then exchanges its ADP for ATP and undergoes a conformational change to form a sliding clamp on DNA. Apparent dissociation constants as determined with SPR are indicated. ( B ) MutS tetramers can bind DNA in each of the two dimers by occasionally bending over. The apparent dissociation constant as determined with SPR of the MutS tetramer for DNA with a GT mismatch is indicated.

    Journal: Nucleic Acids Research

    Article Title: Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation

    doi: 10.1093/nar/gkt582

    Figure Lengend Snippet: States of MutS. ( A ) MutS recognizes a mismatch (pink star) as a dimer. Except when binding homoduplex or a CC mismatch, MutS then exchanges its ADP for ATP and undergoes a conformational change to form a sliding clamp on DNA. Apparent dissociation constants as determined with SPR are indicated. ( B ) MutS tetramers can bind DNA in each of the two dimers by occasionally bending over. The apparent dissociation constant as determined with SPR of the MutS tetramer for DNA with a GT mismatch is indicated.

    Article Snippet: Unless otherwise indicated, DNA for the SPR measurements (obtained from Sigma) contained a 21-base pair long duplex (see Supplementary Table S1 for the full range of DNA sequences measured for MutS binding) with a single-stranded DNA (ssDNA)-overhang consisting of 20 thymidines ((dT)20 ).

    Techniques: Binding Assay, SPR Assay

    Determination of binding constant, kinetics and thermodynamic parameters of the LigBCen2R/GBD interaction by ELISA, SPR, and ITC. (A) Binding of serial concentrations of LigBCen2NR to immobilized GBD by ELISA. Serial concentrations of GST-LigBCen2R, GST-LigBCen2NR, GST-LigBCen2 (positive control), or GST-LigCon (negative control) were added to 1 µM of GBD or BSA coated wells (negative control, data not shown). (B) SPR analysis of LigBCen2R interacting with GBD. 1.5 µM of Recombinant Histidine-tagged LigBCen2R was immobilized on the surface of a Ni-NTA chip. GBD in Tris Buffer containing 100 µM CaCl2 at pH 7.5 flowed through the chip and the concentrations of GBD ranged from 40 to 0.625 µM (from top to bottom). The KD, kon, and koff were obtained from the average of duplicate experiments shown in Table 1 . (C) Determination of the binding affinity by ITC. The cell contained 1 ml of GBD and the syringe contained 250 µl of LigBCen2R (upper panel). Heat differences obtained from 25 injections of LigBCen2R; (lower panel). Integrated curve with experimental data (⋄) and the best fit (-). The thermodynamic parameters are shown as the average of duplicate experiments (KD = 1.88±0.09 µM, ΔH = −29.25±3.58 kcal mol-1, TΔS = −21.47±3.61 kcal mol-1 K-1, n = 0.995±0.01). (D) Fluorescence spectrum of Alexa-488 labeled LigBCen2RW1073C in the presence and absence of GBD. One µM of Alexa-488 labeled LigBCen2RW1073C in Tris buffer was excited at 485 nm. Aliquots of GBD from respective stock solutions were added. The figure shows Alexa488 fluorescence in the presence of 0, 1.62, 3.12, 6.25, 12.5, 25 µM of GBD (Inner plot). The determination of KD of Alexa488 labeled LigBCen2RW1073C and GBD by monitoring the quenching fluorescence intensity of Alexa488 labeled LigBCen2RW1073C titrated by GBD. The emission wavelength recorded in this figure was 513 nm, and KD was revealed by fitting the data point into the equation described in materials and methods (KD = 1.93.4 µM).

    Journal: PLoS ONE

    Article Title: The Terminal Immunoglobulin-Like Repeats of LigA and LigB of Leptospira Enhance Their Binding to Gelatin Binding Domain of Fibronectin and Host Cells

    doi: 10.1371/journal.pone.0011301

    Figure Lengend Snippet: Determination of binding constant, kinetics and thermodynamic parameters of the LigBCen2R/GBD interaction by ELISA, SPR, and ITC. (A) Binding of serial concentrations of LigBCen2NR to immobilized GBD by ELISA. Serial concentrations of GST-LigBCen2R, GST-LigBCen2NR, GST-LigBCen2 (positive control), or GST-LigCon (negative control) were added to 1 µM of GBD or BSA coated wells (negative control, data not shown). (B) SPR analysis of LigBCen2R interacting with GBD. 1.5 µM of Recombinant Histidine-tagged LigBCen2R was immobilized on the surface of a Ni-NTA chip. GBD in Tris Buffer containing 100 µM CaCl2 at pH 7.5 flowed through the chip and the concentrations of GBD ranged from 40 to 0.625 µM (from top to bottom). The KD, kon, and koff were obtained from the average of duplicate experiments shown in Table 1 . (C) Determination of the binding affinity by ITC. The cell contained 1 ml of GBD and the syringe contained 250 µl of LigBCen2R (upper panel). Heat differences obtained from 25 injections of LigBCen2R; (lower panel). Integrated curve with experimental data (⋄) and the best fit (-). The thermodynamic parameters are shown as the average of duplicate experiments (KD = 1.88±0.09 µM, ΔH = −29.25±3.58 kcal mol-1, TΔS = −21.47±3.61 kcal mol-1 K-1, n = 0.995±0.01). (D) Fluorescence spectrum of Alexa-488 labeled LigBCen2RW1073C in the presence and absence of GBD. One µM of Alexa-488 labeled LigBCen2RW1073C in Tris buffer was excited at 485 nm. Aliquots of GBD from respective stock solutions were added. The figure shows Alexa488 fluorescence in the presence of 0, 1.62, 3.12, 6.25, 12.5, 25 µM of GBD (Inner plot). The determination of KD of Alexa488 labeled LigBCen2RW1073C and GBD by monitoring the quenching fluorescence intensity of Alexa488 labeled LigBCen2RW1073C titrated by GBD. The emission wavelength recorded in this figure was 513 nm, and KD was revealed by fitting the data point into the equation described in materials and methods (KD = 1.93.4 µM).

    Article Snippet: Surface Plasmon Resonance (SPR) Association and dissociation rate constants for the interaction of Lig proteins and GBD were measured by SPR analysis performed with a Biacore 2000 instrument (GE Healthcare) at 25°C.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Positive Control, Negative Control, Recombinant, Chromatin Immunoprecipitation, Fluorescence, Labeling

    BIAcore quantification of p55 interaction with peptides encoded by exons 5 and 10. The Surface Plasmon Resonance (SPR) technique was used to quantify the interaction of p55 with the FERM domain of protein 4.1R with and without exon 5-peptide (A–C). The same technique was used to demonstrate the non-competition of exon 5 and exon 10 peptide-binding sites to His-p55 (D). Sensograms were obtained from SPR analysis of the interaction between His-p55 and the FERM domain constructs of protein 4.1R. Recombinant His-p55 protein was immobilized on the CM5 sensor chip and the MBP-FERM domain fusion proteins were injected as an analyte. The wild type His-p55 protein was expressed either in the bacterial cytoplasm or in Sf9 cells with no effect on its binding properties. (A) The binding properties of MBP-FERM and MBP-FERMΔExon 5 fusion proteins to the immobilized His-p55 were measured at 100 nM concentration of each analyte protein. Sensogram of 100 nM MBP (analyte) binding to His-p55 on the chip was used as a negative control. The sensorgrams were generated using a 30 µl/min analyte flow rate (BIAcore 1000 instrument) and included a 3-minute association and a 5-minute dissociation section. (B) Sensograms of the MBP-FERM domain fusion protein (analyte) injected at various concentrations ranging from 50–400 nM. MBP alone at 500 nM was used as a negative control. The flow rate was 30 µl/min over the immobilized His-p55 surface yielding a k D value of 70 nM. (C) Sensograms of the MBP-FERMΔExon 5 domain fusion protein (analyte) injected at various concentrations ranging from 50–400 nM. The MBP at 500 nM was used as a negative control. The flow rate was 30 µl/min over the immobilized His-p55 surface yielding a k D value of 132 nM. (D) The MBP-exon 5 fusion protein (1 µM) was injected over the His-p55 surface, followed by the washing of the chip surface with the running buffer for 25 min at 5 µl/min flow rate. This step was repeated three times, followed by the injection of 1.0 µM MBP-exon 10 for 25 minutes and washing of the chip surface with the running buffer for 25 minutes. The sensograms show that the exon 10-peptide can bind to the p55 surface that is already saturated with the exon 5-peptide.

    Journal: Biochimica et biophysica acta

    Article Title: Alternatively spliced exon 5 of the FERM domain of Protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells

    doi: 10.1016/j.bbamcr.2008.09.012

    Figure Lengend Snippet: BIAcore quantification of p55 interaction with peptides encoded by exons 5 and 10. The Surface Plasmon Resonance (SPR) technique was used to quantify the interaction of p55 with the FERM domain of protein 4.1R with and without exon 5-peptide (A–C). The same technique was used to demonstrate the non-competition of exon 5 and exon 10 peptide-binding sites to His-p55 (D). Sensograms were obtained from SPR analysis of the interaction between His-p55 and the FERM domain constructs of protein 4.1R. Recombinant His-p55 protein was immobilized on the CM5 sensor chip and the MBP-FERM domain fusion proteins were injected as an analyte. The wild type His-p55 protein was expressed either in the bacterial cytoplasm or in Sf9 cells with no effect on its binding properties. (A) The binding properties of MBP-FERM and MBP-FERMΔExon 5 fusion proteins to the immobilized His-p55 were measured at 100 nM concentration of each analyte protein. Sensogram of 100 nM MBP (analyte) binding to His-p55 on the chip was used as a negative control. The sensorgrams were generated using a 30 µl/min analyte flow rate (BIAcore 1000 instrument) and included a 3-minute association and a 5-minute dissociation section. (B) Sensograms of the MBP-FERM domain fusion protein (analyte) injected at various concentrations ranging from 50–400 nM. MBP alone at 500 nM was used as a negative control. The flow rate was 30 µl/min over the immobilized His-p55 surface yielding a k D value of 70 nM. (C) Sensograms of the MBP-FERMΔExon 5 domain fusion protein (analyte) injected at various concentrations ranging from 50–400 nM. The MBP at 500 nM was used as a negative control. The flow rate was 30 µl/min over the immobilized His-p55 surface yielding a k D value of 132 nM. (D) The MBP-exon 5 fusion protein (1 µM) was injected over the His-p55 surface, followed by the washing of the chip surface with the running buffer for 25 min at 5 µl/min flow rate. This step was repeated three times, followed by the injection of 1.0 µM MBP-exon 10 for 25 minutes and washing of the chip surface with the running buffer for 25 minutes. The sensograms show that the exon 10-peptide can bind to the p55 surface that is already saturated with the exon 5-peptide.

    Article Snippet: To quantify these interactions, His-p55 was immobilized on the CM5 sensor chip and the binding of MBP fusion peptides encoded by exons 5 and 10 was measured using the BIAcore SPR assay.

    Techniques: SPR Assay, Binding Assay, Construct, Recombinant, Chromatin Immunoprecipitation, Injection, Concentration Assay, Negative Control, Generated, Flow Cytometry

    AL2-PE38KDEL adaptor-toxin fusion protein and SPR measurements of AL2-PE38KDEL/scFv interaction. ( A ) The AL2-PE38KDEL adaptor-toxin fusion protein has one additional AL fragment linked by a 5-residue linker (Linker2) to the N-terminus of the AL1-PE38KDEL as shown in Fig. 2A . ( B ) SPR measurements of immobilized AL2-PE38KDEL interacting with soluble scFv. The description is the same as in Fig. 2B . The overlaying black lines are the global fit to the experimental data with K d = 5.52 ± 0.03 × 10 −9 M; k on = 3.60 ± 0.06 × 10 5 M −1 S −1 and k off = 1.99 ± 0.04 × 10 −3 S −1 .

    Journal: Scientific Reports

    Article Title: High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries

    doi: 10.1038/srep31878

    Figure Lengend Snippet: AL2-PE38KDEL adaptor-toxin fusion protein and SPR measurements of AL2-PE38KDEL/scFv interaction. ( A ) The AL2-PE38KDEL adaptor-toxin fusion protein has one additional AL fragment linked by a 5-residue linker (Linker2) to the N-terminus of the AL1-PE38KDEL as shown in Fig. 2A . ( B ) SPR measurements of immobilized AL2-PE38KDEL interacting with soluble scFv. The description is the same as in Fig. 2B . The overlaying black lines are the global fit to the experimental data with K d = 5.52 ± 0.03 × 10 −9 M; k on = 3.60 ± 0.06 × 10 5 M −1 S −1 and k off = 1.99 ± 0.04 × 10 −3 S −1 .

    Article Snippet: Surface plasmon resonance (SPR) measurement of binding kinetics of scFv to AL1-PE38KDEL and AL2-PE38KDEL BIAcore T200 (GE Healthcare) instrument was used to determine the binding affinities and kinetics parameters between scFv and AL1-/AL2-PE38KDEL.

    Techniques: SPR Assay

    AL1-PE38KDEL adaptor-toxin fusion protein and SPR measurements of AL1-PE38KDEL/scFv interaction. ( A ) The structure of the trastuzumab scFv variable domain (VH in green and VL in cyan) in complex with protein A (orange) and protein L (blue) is derived from PDB code: 4HKZ. The distance between the C -terminal residue of the VL domain and the N -terminal residue of the VH domain is shown by the red dotted line; the distance between the C -terminal residue of the Protein A and the N -terminal residue of the protein L is shown by the black dotted line. The PE38KDEL, a truncated form of Pseudomonas exotoxin (PDB code: 1IKQ), is colored in red and pink for domain II and III respectively. ( B ) SPR measurements of immobilized AL1-PE38KDEL passed over with 1, 2, 4, 8, 16, 32 and 64 nM Transtuzumab scFv are shown with representative binding traces in colors; the overlaying black lines are the global fit to the experimental data with K d = 5.64 ± 0.03 × 10 −9 M; k on = 4.54 ± 0.06 × 10 5 M −1 S −1 and k off = 2.56 ± 0.04 × 10 −3 S −1 (see Methods).

    Journal: Scientific Reports

    Article Title: High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries

    doi: 10.1038/srep31878

    Figure Lengend Snippet: AL1-PE38KDEL adaptor-toxin fusion protein and SPR measurements of AL1-PE38KDEL/scFv interaction. ( A ) The structure of the trastuzumab scFv variable domain (VH in green and VL in cyan) in complex with protein A (orange) and protein L (blue) is derived from PDB code: 4HKZ. The distance between the C -terminal residue of the VL domain and the N -terminal residue of the VH domain is shown by the red dotted line; the distance between the C -terminal residue of the Protein A and the N -terminal residue of the protein L is shown by the black dotted line. The PE38KDEL, a truncated form of Pseudomonas exotoxin (PDB code: 1IKQ), is colored in red and pink for domain II and III respectively. ( B ) SPR measurements of immobilized AL1-PE38KDEL passed over with 1, 2, 4, 8, 16, 32 and 64 nM Transtuzumab scFv are shown with representative binding traces in colors; the overlaying black lines are the global fit to the experimental data with K d = 5.64 ± 0.03 × 10 −9 M; k on = 4.54 ± 0.06 × 10 5 M −1 S −1 and k off = 2.56 ± 0.04 × 10 −3 S −1 (see Methods).

    Article Snippet: Surface plasmon resonance (SPR) measurement of binding kinetics of scFv to AL1-PE38KDEL and AL2-PE38KDEL BIAcore T200 (GE Healthcare) instrument was used to determine the binding affinities and kinetics parameters between scFv and AL1-/AL2-PE38KDEL.

    Techniques: SPR Assay, Derivative Assay, Binding Assay

    Average time-dependent serum concentrations of GNCgzk scFv-Fc determined by SPR (mean ± SEM; n = 9). Half-life on the GNCgzk scFV-Fc was determined to be 3.9 days.

    Journal: Bioorganic & medicinal chemistry letters

    Article Title: Studies Towards the Improvement of an Anti-Cocaine Monoclonal Antibody for Treatment of Acute Overdose

    doi: 10.1016/j.bmcl.2016.08.081

    Figure Lengend Snippet: Average time-dependent serum concentrations of GNCgzk scFv-Fc determined by SPR (mean ± SEM; n = 9). Half-life on the GNCgzk scFV-Fc was determined to be 3.9 days.

    Article Snippet: The concentration of scFv-Fc retained in the blood was measured by surface plasmon resonance (SPR) using a Biacore 3000 instrument (GE Healthcare) equipped with a research-grade CM5 sensor chip.

    Techniques: SPR Assay

    DNA affinity analyses of dimeric Chro complexes chelated with various divalent metal ions. ( A ) Sensorgrams of the interaction between an immobilized hairpin duplex and the target (5 µM) in a 20 mM Tris–HCl buffer (pH 7.3) containing 50 mM NaCl at 20°C. ( B ) Numerical values of the SPR-derived association equilibrium constants, K a , for immobilized hairpin DNA upon the binding of drugs.

    Journal: PLoS ONE

    Article Title: The Crucial Role of Divalent Metal Ions in the DNA-Acting Efficacy and Inhibition of the Transcription of Dimeric Chromomycin A3

    doi: 10.1371/journal.pone.0043792

    Figure Lengend Snippet: DNA affinity analyses of dimeric Chro complexes chelated with various divalent metal ions. ( A ) Sensorgrams of the interaction between an immobilized hairpin duplex and the target (5 µM) in a 20 mM Tris–HCl buffer (pH 7.3) containing 50 mM NaCl at 20°C. ( B ) Numerical values of the SPR-derived association equilibrium constants, K a , for immobilized hairpin DNA upon the binding of drugs.

    Article Snippet: DNA binding analysis The affinity, association and dissociation between the drug and the DNA duplexes were measured using a BIAcore 3000 A surface plasmon resonance (SPR) instrument (Pharmacia, Uppsala, Sweden) with a SensorChip SA5 from Pharmacia that monitored changes in the refractive index at the sensor chip's surface.

    Techniques: SPR Assay, Derivative Assay, Binding Assay

    Protein expression and purification and surface plasmon resonance analysis of in vitro interactions. A: Coomassie stained protein gel of purified proteins (1µg each) as indicated. All three proteins were expressed in E coli (methods). B: An SPR sensorgram showing relative responses of injections of CaM kinase II gamma G-2 (a) and C-1(b) association domain, both at 250 nM concentration, over an SCP3-immobilized surface. C: Kinetic analysis of serial dilutions (250– 0.3 nM) of CaMKII gamma G-2 association domain binding to SCP3. The lines depict the relative responses of each injection (a-125nM; b-62nM; c-31nM and d-15nM of G-2 association domain) with the lines show the result of global curve-fitting of both ka and kd for three concentrations (a-125nM; b-62nM and c-31nM) using a 1:1 Langmuir binding model with the BIAevalution 4.1 software.

    Journal: The Biochemical journal

    Article Title: Regulation of CaM kinase II by a small CTD phosphatase

    doi: 10.1042/BJ20071582

    Figure Lengend Snippet: Protein expression and purification and surface plasmon resonance analysis of in vitro interactions. A: Coomassie stained protein gel of purified proteins (1µg each) as indicated. All three proteins were expressed in E coli (methods). B: An SPR sensorgram showing relative responses of injections of CaM kinase II gamma G-2 (a) and C-1(b) association domain, both at 250 nM concentration, over an SCP3-immobilized surface. C: Kinetic analysis of serial dilutions (250– 0.3 nM) of CaMKII gamma G-2 association domain binding to SCP3. The lines depict the relative responses of each injection (a-125nM; b-62nM; c-31nM and d-15nM of G-2 association domain) with the lines show the result of global curve-fitting of both ka and kd for three concentrations (a-125nM; b-62nM and c-31nM) using a 1:1 Langmuir binding model with the BIAevalution 4.1 software.

    Article Snippet: The affinities and kinetics of the molecular interactions between SCP3 and the association domains of CaMKII gamma G-2 and C-1 were measured by surface plasmon resonance (SPR) analysis using a Biacore 300 instrument (Biacore, Piscataway, NJ).

    Techniques: Expressing, Purification, SPR Assay, In Vitro, Staining, Chick Chorioallantoic Membrane Assay, Concentration Assay, Binding Assay, Injection, Software

    Evaluation of the role MAb Fc on beneficial immunomodulation. (A) BIAcore SPR analysis of S. mutans adherence to SAG in the presence of sera from BALB/c mice immunized with ICs containing high (saturating) or low (0.1× sub-saturating) concentrations of MAb 6-11A F(ab) 2 or MAb 5-5D F(ab) 2 compared to mice immunized with S. mutans alone. (B) MAb 1-6F competition ELISA. The percent competition by sera from mice immunized with S. mutans alone (white bars), or IC containing MAb 6-11A F(ab) 2 or MAb 5-5D F(ab) 2 at the high saturating (black bars) or low 0.1× sub-saturating concentration (grey bars) is indicated. Results are expressed as mean ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values (C) Quantitative anti-NR21 IgG subclass ELISA. The levels of NR21-specific IgG1, IgG2a, and IgG2b subclass antibodies measured in the sera collected from mice immunized with S. mutans alone (diamonds), or with IC containing MAb 6-11A F(ab) 2 or MAb 5-5D F(ab) 2 at the high saturating (squares) or low 0.1× sub-saturating concentration (triangles) is indicated. Results are expressed as mean ± SEM. Data are representative of at least three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Beneficial Immunomodulation by Streptococcus mutans anti-P1 Monoclonal Antibodies is Fc-independent and Correlates with Increased Exposure of a Relevant Target Epitope

    doi: 10.4049/jimmunol.0803300

    Figure Lengend Snippet: Evaluation of the role MAb Fc on beneficial immunomodulation. (A) BIAcore SPR analysis of S. mutans adherence to SAG in the presence of sera from BALB/c mice immunized with ICs containing high (saturating) or low (0.1× sub-saturating) concentrations of MAb 6-11A F(ab) 2 or MAb 5-5D F(ab) 2 compared to mice immunized with S. mutans alone. (B) MAb 1-6F competition ELISA. The percent competition by sera from mice immunized with S. mutans alone (white bars), or IC containing MAb 6-11A F(ab) 2 or MAb 5-5D F(ab) 2 at the high saturating (black bars) or low 0.1× sub-saturating concentration (grey bars) is indicated. Results are expressed as mean ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values (C) Quantitative anti-NR21 IgG subclass ELISA. The levels of NR21-specific IgG1, IgG2a, and IgG2b subclass antibodies measured in the sera collected from mice immunized with S. mutans alone (diamonds), or with IC containing MAb 6-11A F(ab) 2 or MAb 5-5D F(ab) 2 at the high saturating (squares) or low 0.1× sub-saturating concentration (triangles) is indicated. Results are expressed as mean ± SEM. Data are representative of at least three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.

    Article Snippet: Differences in the immune response include changes in the ability of sera from IC-immunized mice to inhibit S. mutans adherence to SAG, measured using a whole cell BIAcore surface plasmon resonance (SPR) assay, as well as changes in the specificity and isotype of anti-P1 antibodies elicited in mice receiving IC compared to S. mutans alone.

    Techniques: SPR Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Evaluation of immunogenic properties of recombinant P1 polypeptides. (A) BIAcore SPR analysis of S. mutans adherence to salivary agglutinin. Sensograms show the binding of S. mutans to immobilized SAG in the presence of sera collected from sham-immunized (PBS) BALB/c mice compared to those immunized with GC14 (full-length recombinant P1) or with truncated polypeptides NR21, CK1 and CK2. (B) Sera collected from mice immunized with CG14 (filled diamonds), NR21 (open squares), CK1 (filled triangles), CK2 (filled squares), MBP alone (open diamonds), or acrylamide alone (open triangles) were tested for their total IgG reactivity against S. mutans whole cells by ELISA. (C) MAb 1-6F competition ELISA. The percent inhibition of biotin-labeled 1-6F to S. mutans whole cells by sera from mice immunized with CG14 (filled diamonds), NR21 (open squares), CK1 (filled triangles), CK2 (filled squares), MBP alone (open diamonds), or acrylamide alone (open triangles) are shown by line graph. The standard deviation between replicate samples was less than 5 percent in all cases.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Beneficial Immunomodulation by Streptococcus mutans anti-P1 Monoclonal Antibodies is Fc-independent and Correlates with Increased Exposure of a Relevant Target Epitope

    doi: 10.4049/jimmunol.0803300

    Figure Lengend Snippet: Evaluation of immunogenic properties of recombinant P1 polypeptides. (A) BIAcore SPR analysis of S. mutans adherence to salivary agglutinin. Sensograms show the binding of S. mutans to immobilized SAG in the presence of sera collected from sham-immunized (PBS) BALB/c mice compared to those immunized with GC14 (full-length recombinant P1) or with truncated polypeptides NR21, CK1 and CK2. (B) Sera collected from mice immunized with CG14 (filled diamonds), NR21 (open squares), CK1 (filled triangles), CK2 (filled squares), MBP alone (open diamonds), or acrylamide alone (open triangles) were tested for their total IgG reactivity against S. mutans whole cells by ELISA. (C) MAb 1-6F competition ELISA. The percent inhibition of biotin-labeled 1-6F to S. mutans whole cells by sera from mice immunized with CG14 (filled diamonds), NR21 (open squares), CK1 (filled triangles), CK2 (filled squares), MBP alone (open diamonds), or acrylamide alone (open triangles) are shown by line graph. The standard deviation between replicate samples was less than 5 percent in all cases.

    Article Snippet: Differences in the immune response include changes in the ability of sera from IC-immunized mice to inhibit S. mutans adherence to SAG, measured using a whole cell BIAcore surface plasmon resonance (SPR) assay, as well as changes in the specificity and isotype of anti-P1 antibodies elicited in mice receiving IC compared to S. mutans alone.

    Techniques: Recombinant, SPR Assay, Binding Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Labeling, Standard Deviation

    Host genetic background affects beneficial immunomodulation. (A) BIAcore SPR analysis of S. mutans adherence to immobilized salivary agglutinin. Sensograms show the binding of S. mutans to immobilized SAG in the presence of serum from BALB/c or C57/BL6 mice immunized with S. mutans alone or with IC containing MAbs 6-11A or 5-5D at high (saturating) or low (0.1× sub-saturating) concentrations. (B) Evaluation of IgG subclasses reactive with anti- S. mutans whole cells. Sera collected 7 days after primary immunization from BALB/c or C57/BL6 mice immunized with S. mutans alone (diamonds), IC containing MAb 6-11A at the higher concentration (squares), or lower concentration (triangles) were evaluated by quantitative ELISA. (C) Same as panel B except that MAb 5-5D was used in the experiment. (D) A competition ELISA was used to determine the level of MAb 1-6F-like Abs in the serum of BALB/c and C57/BL6 mice immunized with ICs of MAb 6-11A and 5-5D compared to S. mutans alone. The percent inhibition of binding of biotin-labeled 1-6F to S. mutans by sera from mice immunized with bacteria alone (white bars), high MAb concentration IC (black bars), and low MAb concentration IC (grey bars) is indicated. Results are expressed as mean ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values. Data are representative of at least three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Beneficial Immunomodulation by Streptococcus mutans anti-P1 Monoclonal Antibodies is Fc-independent and Correlates with Increased Exposure of a Relevant Target Epitope

    doi: 10.4049/jimmunol.0803300

    Figure Lengend Snippet: Host genetic background affects beneficial immunomodulation. (A) BIAcore SPR analysis of S. mutans adherence to immobilized salivary agglutinin. Sensograms show the binding of S. mutans to immobilized SAG in the presence of serum from BALB/c or C57/BL6 mice immunized with S. mutans alone or with IC containing MAbs 6-11A or 5-5D at high (saturating) or low (0.1× sub-saturating) concentrations. (B) Evaluation of IgG subclasses reactive with anti- S. mutans whole cells. Sera collected 7 days after primary immunization from BALB/c or C57/BL6 mice immunized with S. mutans alone (diamonds), IC containing MAb 6-11A at the higher concentration (squares), or lower concentration (triangles) were evaluated by quantitative ELISA. (C) Same as panel B except that MAb 5-5D was used in the experiment. (D) A competition ELISA was used to determine the level of MAb 1-6F-like Abs in the serum of BALB/c and C57/BL6 mice immunized with ICs of MAb 6-11A and 5-5D compared to S. mutans alone. The percent inhibition of binding of biotin-labeled 1-6F to S. mutans by sera from mice immunized with bacteria alone (white bars), high MAb concentration IC (black bars), and low MAb concentration IC (grey bars) is indicated. Results are expressed as mean ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values. Data are representative of at least three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.

    Article Snippet: Differences in the immune response include changes in the ability of sera from IC-immunized mice to inhibit S. mutans adherence to SAG, measured using a whole cell BIAcore surface plasmon resonance (SPR) assay, as well as changes in the specificity and isotype of anti-P1 antibodies elicited in mice receiving IC compared to S. mutans alone.

    Techniques: SPR Assay, Binding Assay, Mouse Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Labeling

    Evaluation of the role of activating Fc receptors in beneficial immunomodulation by anti-P1 MAbs. (A) BIAcore SPR analysis of S. mutans adherence to immobilized salivary agglutinin. Sensograms show the binding of S. mutans to immobilized SAG in the presence of serum from Fcer1g transgenic mice immunized with S. mutans alone or with IC containing high (saturating) or low (0.1× sub-saturating) concentrations of MAb 5-5D. (B) MAb 1-6F competition ELISA. The percent competition by sera from mice immunized with S. mutans alone (white bars), IC containing the high (saturating) MAb concentration (black bars), or IC containing the low (0.1× sub-saturating) MAb concentration (grey bars) is indicated. Results are expressed as mean ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values. (C) Quantitative anti-NR21 IgG subclass ELISA. Levels of anti-NR21 specific IgG1, IgG2a, and IgG2b subclass antibodies were measured in sera collected from Fcer1g transgenic mice immunized with S. mutans alone (diamonds), or with IC containing MAb 5-5D at the high saturating (squares) or low 0.1× sub-saturating concentration (triangles). Results are expressed as mean ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values. Data are representative of at least three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Beneficial Immunomodulation by Streptococcus mutans anti-P1 Monoclonal Antibodies is Fc-independent and Correlates with Increased Exposure of a Relevant Target Epitope

    doi: 10.4049/jimmunol.0803300

    Figure Lengend Snippet: Evaluation of the role of activating Fc receptors in beneficial immunomodulation by anti-P1 MAbs. (A) BIAcore SPR analysis of S. mutans adherence to immobilized salivary agglutinin. Sensograms show the binding of S. mutans to immobilized SAG in the presence of serum from Fcer1g transgenic mice immunized with S. mutans alone or with IC containing high (saturating) or low (0.1× sub-saturating) concentrations of MAb 5-5D. (B) MAb 1-6F competition ELISA. The percent competition by sera from mice immunized with S. mutans alone (white bars), IC containing the high (saturating) MAb concentration (black bars), or IC containing the low (0.1× sub-saturating) MAb concentration (grey bars) is indicated. Results are expressed as mean ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values. (C) Quantitative anti-NR21 IgG subclass ELISA. Levels of anti-NR21 specific IgG1, IgG2a, and IgG2b subclass antibodies were measured in sera collected from Fcer1g transgenic mice immunized with S. mutans alone (diamonds), or with IC containing MAb 5-5D at the high saturating (squares) or low 0.1× sub-saturating concentration (triangles). Results are expressed as mean ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values. Data are representative of at least three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.

    Article Snippet: Differences in the immune response include changes in the ability of sera from IC-immunized mice to inhibit S. mutans adherence to SAG, measured using a whole cell BIAcore surface plasmon resonance (SPR) assay, as well as changes in the specificity and isotype of anti-P1 antibodies elicited in mice receiving IC compared to S. mutans alone.

    Techniques: SPR Assay, Binding Assay, Transgenic Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Re-evaluation of the immunomodulatory properties of MAb 4-10A. (A) BIAcore SPR analysis of S. mutans adherence to salivary agglutinin in the presence of sera collected from BALB/c mice immunized with S. mutans alone or with ICs containing MAb 4-10A at the indicated dilutions. For the sake of clarity the change in resonance units detected over the 60 second injection cycle rather than the complete sensograms are shown. (B) MAb 1-6F competition ELISA. Sera collected from mice 7 days after primary immunization with S. mutans alone or with ICs containing MAb 4-10A at the indicated dilutions were tested for their ability to inhibit binding of biotin-labeled 1-6F to S. mutans whole cells. Results are expressed as the mean percent inhibition ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values. (C) Quantitative anti-NR21 IgG subclass ELISA. The levels of NR21-specific IgG1, IgG2a, and IgG2b subclass antibodies were measured in the same sera described in panel B. Results are expressed as mean ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values. (D) MAb 4-10A enhancement of MAb 1-6F binding to S. mutans whole cells by ELISA. The percent increase or decrease in binding of biotin-labeled MAb 1-6F to S. mutans in the presence of MAb 4-10A added at the indicated dilution was calculated. Data are representative of three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Beneficial Immunomodulation by Streptococcus mutans anti-P1 Monoclonal Antibodies is Fc-independent and Correlates with Increased Exposure of a Relevant Target Epitope

    doi: 10.4049/jimmunol.0803300

    Figure Lengend Snippet: Re-evaluation of the immunomodulatory properties of MAb 4-10A. (A) BIAcore SPR analysis of S. mutans adherence to salivary agglutinin in the presence of sera collected from BALB/c mice immunized with S. mutans alone or with ICs containing MAb 4-10A at the indicated dilutions. For the sake of clarity the change in resonance units detected over the 60 second injection cycle rather than the complete sensograms are shown. (B) MAb 1-6F competition ELISA. Sera collected from mice 7 days after primary immunization with S. mutans alone or with ICs containing MAb 4-10A at the indicated dilutions were tested for their ability to inhibit binding of biotin-labeled 1-6F to S. mutans whole cells. Results are expressed as the mean percent inhibition ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values. (C) Quantitative anti-NR21 IgG subclass ELISA. The levels of NR21-specific IgG1, IgG2a, and IgG2b subclass antibodies were measured in the same sera described in panel B. Results are expressed as mean ± SEM and statistical significance compared to serum from S. mutans only immunized mice is indicated by P values. (D) MAb 4-10A enhancement of MAb 1-6F binding to S. mutans whole cells by ELISA. The percent increase or decrease in binding of biotin-labeled MAb 1-6F to S. mutans in the presence of MAb 4-10A added at the indicated dilution was calculated. Data are representative of three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.

    Article Snippet: Differences in the immune response include changes in the ability of sera from IC-immunized mice to inhibit S. mutans adherence to SAG, measured using a whole cell BIAcore surface plasmon resonance (SPR) assay, as well as changes in the specificity and isotype of anti-P1 antibodies elicited in mice receiving IC compared to S. mutans alone.

    Techniques: SPR Assay, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Binding Assay, Labeling, Inhibition

    RAGE binds only to the toxic, prefibrillar form of h-IAPP. ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) SPR shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.

    Journal: The Journal of Clinical Investigation

    Article Title: RAGE binds preamyloid IAPP intermediates and mediates pancreatic β cell proteotoxicity

    doi: 10.1172/JCI85210

    Figure Lengend Snippet: RAGE binds only to the toxic, prefibrillar form of h-IAPP. ( A ) Schematic diagram showing the design of h-IAPP/sRAGE binding experiments. Blue arrows indicate the time points at which sRAGE was added to h-IAPP over the course of amyloid formation. ( B ) In the sRAGE Trp fluorescence assays, a 1:1 molar addition of sRAGE to h-IAPP (blue circles) led to a wave of fluorescence quenching that mirrored the wave of h-IAPP toxicity shown in D . No change in fluorescence was observed for sRAGE alone (black squares) or with a 1:1 molar addition of sRAGE to r-IAPP (white triangles). h-IAPP, in the absence of sRAGE (red circles), and r-IAPP, in the absence of sRAGE (green triangles), were used as nonfluorescent controls. ( C ) Thioflavin-T–binding assays, carried out concurrently with sRAGE Trp fluorescence assays and β cell metabolic assays, monitored the kinetics of amyloid formation (25°C) in the peptide solutions used in the experiments shown in B and D . h-IAPP (red circles) and r-IAPP (green triangles). ( D ) Time-resolved Alamar Blue metabolic assays in INS-1 β cells treated with h-IAPP (red circles) or r-IAPP (green triangles) demonstrated that LP intermediates were the most toxic form of h-IAPP. ( E ) SPR shows that sRAGE bound h-IAPP LP intermediates (blue line) but not t 0 species (black dashed line) or SP amyloid fibrils (red dashed line). In B – D , the symbol (§) indicates the time point at which the maximum sRAGE Trp fluorescence quenching was observed. The final peptide concentration after transferring peptide aliquots into β cell assays was 14 μM. Data are representative of 3 to 10 independent experiments. Data in C and D represent the mean ± SD of a minimum of 3 to 6 technical replicates per time point. Error bars for some data points are smaller than the size of the symbols.

    Article Snippet: In SPR studies, sRAGE was immobilized on the sensor chip, and the interaction of different h-IAPP species with sRAGE was measured using a GE Healthcare SPR instrument.

    Techniques: Binding Assay, Fluorescence, SPR Assay, Concentration Assay, Transferring