mouse cdh23  (New England Biolabs)


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    Name:
    pTXB1 Vector DNA
    Description:
    pTXB1 Vector DNA 10 ug
    Catalog Number:
    n6707s
    Price:
    133
    Size:
    10 ug
    Category:
    E coli Expression Vectors
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    Structured Review

    New England Biolabs mouse cdh23
    pTXB1 Vector DNA
    pTXB1 Vector DNA 10 ug
    https://www.bioz.com/result/mouse cdh23/product/New England Biolabs
    Average 91 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    mouse cdh23 - by Bioz Stars, 2020-09
    91/100 stars

    Images

    1) Product Images from "Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins"

    Article Title: Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189546

    Quantitative SPR measurements of cdh23 molecules binding to pcdh15. (A) Left, association and dissociation of the cdh23(WT)-pcdh15(WT) complex. Experimental data (sensorgrams) are represented in a gradient of green to blue colors for different concentrations of cdh23(WT) as labeled. Red lines indicate fitted model parameters (RMSD = 2.89). Injection peaks were removed and not fitted. Top three traces correspond to 5, 10, and 15 μM, respectively, but the 10 μM trace is not labeled for clarity. Black arrow indicates the position of equilibrium SPR signal (R eq ). Middle panel shows the fitting of R eq to a Langmuir binding isotherm at different concentrations of analyte. Measurements for selected concentrations were done in duplicates. Right panel shows a heat map of the k off and K D distribution from the global fit of all traces in corresponding leftmost panel. The signal density of the peaks in the k off and K D distribution plot can directly be discerned from their color, which is scaled according to the color bar on the right side of the distribution plot [ 48 ]. (B) Association and dissociation curves for the T15E-G16D complex shown as in (A) (RMSD = 11.08). Top three traces correspond to 15, 20, and 25 μM, respectively, but the 15 and 20 μM traces are not labeled for clarity. The data were analyzed with the EVILFIT algorithm and the Biacore evaluation software.
    Figure Legend Snippet: Quantitative SPR measurements of cdh23 molecules binding to pcdh15. (A) Left, association and dissociation of the cdh23(WT)-pcdh15(WT) complex. Experimental data (sensorgrams) are represented in a gradient of green to blue colors for different concentrations of cdh23(WT) as labeled. Red lines indicate fitted model parameters (RMSD = 2.89). Injection peaks were removed and not fitted. Top three traces correspond to 5, 10, and 15 μM, respectively, but the 10 μM trace is not labeled for clarity. Black arrow indicates the position of equilibrium SPR signal (R eq ). Middle panel shows the fitting of R eq to a Langmuir binding isotherm at different concentrations of analyte. Measurements for selected concentrations were done in duplicates. Right panel shows a heat map of the k off and K D distribution from the global fit of all traces in corresponding leftmost panel. The signal density of the peaks in the k off and K D distribution plot can directly be discerned from their color, which is scaled according to the color bar on the right side of the distribution plot [ 48 ]. (B) Association and dissociation curves for the T15E-G16D complex shown as in (A) (RMSD = 11.08). Top three traces correspond to 15, 20, and 25 μM, respectively, but the 15 and 20 μM traces are not labeled for clarity. The data were analyzed with the EVILFIT algorithm and the Biacore evaluation software.

    Techniques Used: SPR Assay, Binding Assay, Labeling, Injection, Software

    Heterotetrameric tip link made of CDH23 bound to PCDH15. (A) The tip link is formed by a CDH23 parallel dimer interacting tip-to-tip with a PCDH15 parallel dimer [ 39 ]. These proteins feature 27 and 11 extracellular cadherin (EC) repeats, respectively. (B) Ribbon diagram of mouse cdh23 (blue) bound to pcdh15 (magenta) with Ca 2+ ions as green spheres (PDB ID: 4APX). Sites of deafness-causing mutations R113 and I108 in PCDH15 are shown in stick representation and circled. (C D) Detail of I108 (C) and R113 (D) with surrounding residues in the cdh23 and pcdh15 interface.
    Figure Legend Snippet: Heterotetrameric tip link made of CDH23 bound to PCDH15. (A) The tip link is formed by a CDH23 parallel dimer interacting tip-to-tip with a PCDH15 parallel dimer [ 39 ]. These proteins feature 27 and 11 extracellular cadherin (EC) repeats, respectively. (B) Ribbon diagram of mouse cdh23 (blue) bound to pcdh15 (magenta) with Ca 2+ ions as green spheres (PDB ID: 4APX). Sites of deafness-causing mutations R113 and I108 in PCDH15 are shown in stick representation and circled. (C D) Detail of I108 (C) and R113 (D) with surrounding residues in the cdh23 and pcdh15 interface.

    Techniques Used:

    Variation of ΔT m at increasing ratios of cdh23 and pcdh15. (A) Variation of ΔT m for WT and two deafness-related complexes at increasing concentration ratios. The WT ΔT m increases from ~2 to ~5.5°C from 1:1 to 5:1 ratios. The deafness mutants have ΔT m
    Figure Legend Snippet: Variation of ΔT m at increasing ratios of cdh23 and pcdh15. (A) Variation of ΔT m for WT and two deafness-related complexes at increasing concentration ratios. The WT ΔT m increases from ~2 to ~5.5°C from 1:1 to 5:1 ratios. The deafness mutants have ΔT m

    Techniques Used: Concentration Assay

    Rate and equilibrium constants for different mutants vs ΔΔT m . (A-C) K D vs ΔΔT m (A), k off vs ΔΔT m (B), and k on vs ΔΔT m (C) for various cdh23-pcdh15 complexes (WT-WT: light blue; T15E-WT: red; WT-G16D: yellow; T15E-G16D: light green, F7E-WT: cyan; S76V-WT: navy blue; WT-Q165L: pink). Squares and circles represent data points for ΔΔT m at 1:1 and 5:1 ratios, respectively. The WT-WT complex lies at the origin. Data points along the dashed diagonal line in A support ΔΔT m as a good predictor of K D , while data points off the diagonal indicate exceptions (most notably F7E-WT). Vertical error bars represent standard deviation for measurements of the rate or equilibrium constant of that mutant. Horizontal error bars represent the standard deviation for the ΔT m measurement.
    Figure Legend Snippet: Rate and equilibrium constants for different mutants vs ΔΔT m . (A-C) K D vs ΔΔT m (A), k off vs ΔΔT m (B), and k on vs ΔΔT m (C) for various cdh23-pcdh15 complexes (WT-WT: light blue; T15E-WT: red; WT-G16D: yellow; T15E-G16D: light green, F7E-WT: cyan; S76V-WT: navy blue; WT-Q165L: pink). Squares and circles represent data points for ΔΔT m at 1:1 and 5:1 ratios, respectively. The WT-WT complex lies at the origin. Data points along the dashed diagonal line in A support ΔΔT m as a good predictor of K D , while data points off the diagonal indicate exceptions (most notably F7E-WT). Vertical error bars represent standard deviation for measurements of the rate or equilibrium constant of that mutant. Horizontal error bars represent the standard deviation for the ΔT m measurement.

    Techniques Used: Standard Deviation, Mutagenesis

    Thermal melting curves of cdh23 and pcdh15 variants monitored with SYPRO orange. (A) Upper panels show normalized fluorescence for melting curves with cdh23(WT) in cyan, pcdh15(WT) in magenta, and the mixture in black. Lower panels show the derivative of the normalized melting curve. Concentration ratios from left to right are: 1:1, 2:1, 3:1, 4:1, 5:1 (cdh23:pcdh15). (B-C) Overlay of the melting curves for pcdh15(WT) (B) and for pcdh15(R113G) (C) at indicated cdh23:pcdh15 ratios. The curves were normalized to pcdh15 only (first peak).
    Figure Legend Snippet: Thermal melting curves of cdh23 and pcdh15 variants monitored with SYPRO orange. (A) Upper panels show normalized fluorescence for melting curves with cdh23(WT) in cyan, pcdh15(WT) in magenta, and the mixture in black. Lower panels show the derivative of the normalized melting curve. Concentration ratios from left to right are: 1:1, 2:1, 3:1, 4:1, 5:1 (cdh23:pcdh15). (B-C) Overlay of the melting curves for pcdh15(WT) (B) and for pcdh15(R113G) (C) at indicated cdh23:pcdh15 ratios. The curves were normalized to pcdh15 only (first peak).

    Techniques Used: Fluorescence, Concentration Assay

    Mapping of rationally designed mutation sites on the structure of the cdh23 and pcdh15 complex. (A) Surface representation of cdh23 (blue and cyan) bound to pcdh15 (purple and pink; PDB ID: 4APX). (B) Cdh23 and pcdh15 interaction surfaces exposed with mutation sites labeled. Residues labeled in red are mutated in inherited deafness. Underlined labels indicate sites that belong to paired mutant complexes cdh23(H11K)-pcdh15(Q218E) and cdh23(T79E)-pcdh15(H91R).
    Figure Legend Snippet: Mapping of rationally designed mutation sites on the structure of the cdh23 and pcdh15 complex. (A) Surface representation of cdh23 (blue and cyan) bound to pcdh15 (purple and pink; PDB ID: 4APX). (B) Cdh23 and pcdh15 interaction surfaces exposed with mutation sites labeled. Residues labeled in red are mutated in inherited deafness. Underlined labels indicate sites that belong to paired mutant complexes cdh23(H11K)-pcdh15(Q218E) and cdh23(T79E)-pcdh15(H91R).

    Techniques Used: Mutagenesis, Labeling

    2) Product Images from "Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides"

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides

    Journal: Biopolymers

    doi: 10.1002/bip.21391

    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Figure Legend Snippet: Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Techniques Used: Sequencing, Plasmid Preparation, Expressing, Binding Assay, Affinity Purification, Recombinant, Ligation

    3) Product Images from "Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides"

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides

    Journal: Biopolymers

    doi: 10.1002/bip.21391

    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Figure Legend Snippet: Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Techniques Used: Sequencing, Plasmid Preparation, Expressing, Binding Assay, Affinity Purification, Recombinant, Ligation

    4) Product Images from "Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides"

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides

    Journal: Biopolymers

    doi: 10.1002/bip.21391

    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Figure Legend Snippet: Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Techniques Used: Sequencing, Plasmid Preparation, Expressing, Binding Assay, Affinity Purification, Recombinant, Ligation

    5) Product Images from "Fidelity Index Determination of DNA Methyltransferases"

    Article Title: Fidelity Index Determination of DNA Methyltransferases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063866

    SDS-PAGE of purified methyltransferases. Lane M is the ColorPlus protein ladder (NEB # P7710). Lane 1: M.MspI, Lane 2: M.AluI, Lane 3: M.EcoRI, Lane 4: M.EcoKDam, Lane 5: M.HpaII, and Lane 6: M.HaeIII.
    Figure Legend Snippet: SDS-PAGE of purified methyltransferases. Lane M is the ColorPlus protein ladder (NEB # P7710). Lane 1: M.MspI, Lane 2: M.AluI, Lane 3: M.EcoRI, Lane 4: M.EcoKDam, Lane 5: M.HpaII, and Lane 6: M.HaeIII.

    Techniques Used: SDS Page, Purification

    6) Product Images from "Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides"

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides

    Journal: Biopolymers

    doi: 10.1002/bip.21391

    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Figure Legend Snippet: Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Techniques Used: Sequencing, Plasmid Preparation, Expressing, Binding Assay, Affinity Purification, Recombinant, Ligation

    7) Product Images from "Facile Method for the Site-Specific, Covalent Attachment of full-length IgG onto Nanoparticles"

    Article Title: Facile Method for the Site-Specific, Covalent Attachment of full-length IgG onto Nanoparticles

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    doi: 10.1002/smll.201303629

    SDS-PAGE with Coomassie staining confirming the in vivo incorporation of BPA into expressed Protein Z T7 competent E. coli were co-transformed with the pEVOL-pBpf plasmid containing the amber suppressor tRNA/aminoacyl transferase pair and the pTXB1 plasmid, which codes for Protein Z with an amber codon mutation (ProZ F13BPA). Following induction of protein expression, cell lysates, with or without BPA in the media, were evaluated by SDS-PAGE stained with Coomassie (lanes 1 and 2, respectively). Analogous studies were performed with E. coli that express wild-type Protein Z (lane 3) and unmodified T7 competent cells (lane 4).
    Figure Legend Snippet: SDS-PAGE with Coomassie staining confirming the in vivo incorporation of BPA into expressed Protein Z T7 competent E. coli were co-transformed with the pEVOL-pBpf plasmid containing the amber suppressor tRNA/aminoacyl transferase pair and the pTXB1 plasmid, which codes for Protein Z with an amber codon mutation (ProZ F13BPA). Following induction of protein expression, cell lysates, with or without BPA in the media, were evaluated by SDS-PAGE stained with Coomassie (lanes 1 and 2, respectively). Analogous studies were performed with E. coli that express wild-type Protein Z (lane 3) and unmodified T7 competent cells (lane 4).

    Techniques Used: SDS Page, Staining, In Vivo, Transformation Assay, Plasmid Preparation, Mutagenesis, Expressing

    8) Product Images from "Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau"

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-018-0562-9

    Inhibition of in-vitro aggregation of pS422-tau by dmCBTAU-22.1. a Schematic representation of the chemical ligation process applied to preparation pS422-tau. Ser 422 (red) is phosphorylated in the CBTAU-22.1 epitope and Ser 416 (blue) is mutated into Cys as a result of the chemical ligation process. b SDS-PAGE analysis of the ligation reaction progress. Lane 1 corresponds to the full tau1–415-Mxe-CBD multidomain protein produced in E. coli after cloning into the pTXB1 vector. For the purposes of this analysis the protein was purified via His-tag affinity chromatography. Lane 2 corresponds to the thioester product obtained by treatment of the Lane 1 product with excess MESNa. Lane 3 represents the ligation product obtained by reaction of the Lane 2 product with tau peptide CL22D-P. c Size Exclusion Chromatography profiles of pS422-tau (green) and non-phosphorylated 2N4R-tau (blue). d Western blot of pS422-tau and non-phosphorylated 2N4R-tau with dmCBTAU-22.1. e Aggregation of pS422-tau in the absence (black) or presence of dmCBTAU-27.1 (red) or dmCBTAU-22.1 (blue) as monitored continuously by ThT fluorescence. The molar ratio between pS422-tau and IgG was 1: 0.6 in both cases. Each experimental condition was tested in three independent replicates, the red triplicates and two of the blue triplicates overlap and cannot be visually distinguished. f Atomic force microscopy of pS422-tau fibrils. The two panels correspond to AFM images with sizes of 6 × 6 μm and 1.5 × 1.5 μm, respectively
    Figure Legend Snippet: Inhibition of in-vitro aggregation of pS422-tau by dmCBTAU-22.1. a Schematic representation of the chemical ligation process applied to preparation pS422-tau. Ser 422 (red) is phosphorylated in the CBTAU-22.1 epitope and Ser 416 (blue) is mutated into Cys as a result of the chemical ligation process. b SDS-PAGE analysis of the ligation reaction progress. Lane 1 corresponds to the full tau1–415-Mxe-CBD multidomain protein produced in E. coli after cloning into the pTXB1 vector. For the purposes of this analysis the protein was purified via His-tag affinity chromatography. Lane 2 corresponds to the thioester product obtained by treatment of the Lane 1 product with excess MESNa. Lane 3 represents the ligation product obtained by reaction of the Lane 2 product with tau peptide CL22D-P. c Size Exclusion Chromatography profiles of pS422-tau (green) and non-phosphorylated 2N4R-tau (blue). d Western blot of pS422-tau and non-phosphorylated 2N4R-tau with dmCBTAU-22.1. e Aggregation of pS422-tau in the absence (black) or presence of dmCBTAU-27.1 (red) or dmCBTAU-22.1 (blue) as monitored continuously by ThT fluorescence. The molar ratio between pS422-tau and IgG was 1: 0.6 in both cases. Each experimental condition was tested in three independent replicates, the red triplicates and two of the blue triplicates overlap and cannot be visually distinguished. f Atomic force microscopy of pS422-tau fibrils. The two panels correspond to AFM images with sizes of 6 × 6 μm and 1.5 × 1.5 μm, respectively

    Techniques Used: Inhibition, In Vitro, Ligation, SDS Page, Produced, Clone Assay, Plasmid Preparation, Purification, Affinity Chromatography, Size-exclusion Chromatography, Western Blot, Fluorescence, Microscopy

    9) Product Images from "Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau"

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-018-0562-9

    Inhibition of in-vitro aggregation of pS422-tau by dmCBTAU-22.1. a Schematic representation of the chemical ligation process applied to preparation pS422-tau. Ser 422 (red) is phosphorylated in the CBTAU-22.1 epitope and Ser 416 (blue) is mutated into Cys as a result of the chemical ligation process. b SDS-PAGE analysis of the ligation reaction progress. Lane 1 corresponds to the full tau1–415-Mxe-CBD multidomain protein produced in E. coli after cloning into the pTXB1 vector. For the purposes of this analysis the protein was purified via His-tag affinity chromatography. Lane 2 corresponds to the thioester product obtained by treatment of the Lane 1 product with excess MESNa. Lane 3 represents the ligation product obtained by reaction of the Lane 2 product with tau peptide CL22D-P. c Size Exclusion Chromatography profiles of pS422-tau (green) and non-phosphorylated 2N4R-tau (blue). d Western blot of pS422-tau and non-phosphorylated 2N4R-tau with dmCBTAU-22.1. e Aggregation of pS422-tau in the absence (black) or presence of dmCBTAU-27.1 (red) or dmCBTAU-22.1 (blue) as monitored continuously by ThT fluorescence. The molar ratio between pS422-tau and IgG was 1: 0.6 in both cases. Each experimental condition was tested in three independent replicates, the red triplicates and two of the blue triplicates overlap and cannot be visually distinguished. f Atomic force microscopy of pS422-tau fibrils. The two panels correspond to AFM images with sizes of 6 × 6 μm and 1.5 × 1.5 μm, respectively
    Figure Legend Snippet: Inhibition of in-vitro aggregation of pS422-tau by dmCBTAU-22.1. a Schematic representation of the chemical ligation process applied to preparation pS422-tau. Ser 422 (red) is phosphorylated in the CBTAU-22.1 epitope and Ser 416 (blue) is mutated into Cys as a result of the chemical ligation process. b SDS-PAGE analysis of the ligation reaction progress. Lane 1 corresponds to the full tau1–415-Mxe-CBD multidomain protein produced in E. coli after cloning into the pTXB1 vector. For the purposes of this analysis the protein was purified via His-tag affinity chromatography. Lane 2 corresponds to the thioester product obtained by treatment of the Lane 1 product with excess MESNa. Lane 3 represents the ligation product obtained by reaction of the Lane 2 product with tau peptide CL22D-P. c Size Exclusion Chromatography profiles of pS422-tau (green) and non-phosphorylated 2N4R-tau (blue). d Western blot of pS422-tau and non-phosphorylated 2N4R-tau with dmCBTAU-22.1. e Aggregation of pS422-tau in the absence (black) or presence of dmCBTAU-27.1 (red) or dmCBTAU-22.1 (blue) as monitored continuously by ThT fluorescence. The molar ratio between pS422-tau and IgG was 1: 0.6 in both cases. Each experimental condition was tested in three independent replicates, the red triplicates and two of the blue triplicates overlap and cannot be visually distinguished. f Atomic force microscopy of pS422-tau fibrils. The two panels correspond to AFM images with sizes of 6 × 6 μm and 1.5 × 1.5 μm, respectively

    Techniques Used: Inhibition, In Vitro, Ligation, SDS Page, Produced, Clone Assay, Plasmid Preparation, Purification, Affinity Chromatography, Size-exclusion Chromatography, Western Blot, Fluorescence, Microscopy

    10) Product Images from "Fidelity Index Determination of DNA Methyltransferases"

    Article Title: Fidelity Index Determination of DNA Methyltransferases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063866

    SDS-PAGE of purified methyltransferases. Lane M is the ColorPlus protein ladder (NEB # P7710). Lane 1: M.MspI, Lane 2: M.AluI, Lane 3: M.EcoRI, Lane 4: M.EcoKDam, Lane 5: M.HpaII, and Lane 6: M.HaeIII.
    Figure Legend Snippet: SDS-PAGE of purified methyltransferases. Lane M is the ColorPlus protein ladder (NEB # P7710). Lane 1: M.MspI, Lane 2: M.AluI, Lane 3: M.EcoRI, Lane 4: M.EcoKDam, Lane 5: M.HpaII, and Lane 6: M.HaeIII.

    Techniques Used: SDS Page, Purification

    11) Product Images from "Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides"

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides

    Journal: Biopolymers

    doi: 10.1002/bip.21391

    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Figure Legend Snippet: Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Techniques Used: Sequencing, Plasmid Preparation, Expressing, Binding Assay, Affinity Purification, Recombinant, Ligation

    12) Product Images from "Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides"

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides

    Journal: Biopolymers

    doi: 10.1002/bip.21391

    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Figure Legend Snippet: Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Techniques Used: Sequencing, Plasmid Preparation, Expressing, Binding Assay, Affinity Purification, Recombinant, Ligation

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    Sequencing:

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    Article Snippet: .. bZIP domains were cloned from plasmids pWY16 to pWY19 ( ) or A. nidulans genomic DNA into a modified pTXB1 (NEB) plasmid using the sequence and ligation-independent cloning (SLIC) method , or were restriction-digested with Xho I and Nsi I. .. Proteins were expressed as intein–chitin binding domain fusions in RP3098 E. coli by growing 1 l LB cultures at 37 °C to OD600 0.4–0.8, at which point expression was induced with 0.5 mM IPTG.

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    Article Title: Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites, et al. Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites
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    Article Snippet: .. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707). ..

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    New England Biolabs ptxb1 vector
    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the <t>pTXB1</t> vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Ptxb1 Vector, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Journal: Biopolymers

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides

    doi: 10.1002/bip.21391

    Figure Lengend Snippet: Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Article Snippet: As described above, a miniprep of DNA for the pTXB1 vector containing Aβ1-29 was used as a template for synthesis of the mutated strand by PCR.

    Techniques: Sequencing, Plasmid Preparation, Expressing, Binding Assay, Affinity Purification, Recombinant, Ligation