ptxb1  (New England Biolabs)


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    Name:
    pTXB1 Vector DNA
    Description:
    pTXB1 Vector DNA 10 ug
    Catalog Number:
    N6707S
    Price:
    133
    Category:
    E coli Expression Vectors
    Size:
    10 ug
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    Structured Review

    New England Biolabs ptxb1
    pTXB1 Vector DNA
    pTXB1 Vector DNA 10 ug
    https://www.bioz.com/result/ptxb1/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ptxb1 - by Bioz Stars, 2021-04
    97/100 stars

    Images

    1) Product Images from "The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness"

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness

    Journal: Viruses

    doi: 10.3390/v10070343

    MotB copurifies with H-NS and StpA. An SDS-PAGE gel, stained with Coomassie, is shown for the following samples obtained from BL21(DE3)/pLysE containing either the vector pTXB1 (lanes 3–10) or pTXB1-MotB (lanes 11–18): lysate applied to chitin resin (lanes 3 and 11); column flow through (lanes 4 and 12); resin sample after flow through (lanes 5 and 13); first and second column wash with buffer containing 500 mM NaCl (lanes 6, 7 and 14, 15); resin sample after second 500 mM salt wash (lanes 8 and 16); column wash with buffer containing 1 M NaCl (lanes 9 and 17); final resin sample after 1 M salt wash (lanes 10 and 18). Lane 1 corresponds to SeeBlue Plus2 protein standard; corresponding molecular weights are shown on the left. Bands corresponding to MotB-Intein/CBD and Intein/CBD are indicated. Species indicated with arrows were identified by mass spectrometry. The ~36 kDa protein labeled as “degraded construct” was identified as partially degraded MotB-Intein/CBD. The ~16 kDa protein was identified as H-NS and StpA.
    Figure Legend Snippet: MotB copurifies with H-NS and StpA. An SDS-PAGE gel, stained with Coomassie, is shown for the following samples obtained from BL21(DE3)/pLysE containing either the vector pTXB1 (lanes 3–10) or pTXB1-MotB (lanes 11–18): lysate applied to chitin resin (lanes 3 and 11); column flow through (lanes 4 and 12); resin sample after flow through (lanes 5 and 13); first and second column wash with buffer containing 500 mM NaCl (lanes 6, 7 and 14, 15); resin sample after second 500 mM salt wash (lanes 8 and 16); column wash with buffer containing 1 M NaCl (lanes 9 and 17); final resin sample after 1 M salt wash (lanes 10 and 18). Lane 1 corresponds to SeeBlue Plus2 protein standard; corresponding molecular weights are shown on the left. Bands corresponding to MotB-Intein/CBD and Intein/CBD are indicated. Species indicated with arrows were identified by mass spectrometry. The ~36 kDa protein labeled as “degraded construct” was identified as partially degraded MotB-Intein/CBD. The ~16 kDa protein was identified as H-NS and StpA.

    Techniques Used: SDS Page, Staining, Plasmid Preparation, Flow Cytometry, Mass Spectrometry, Labeling, Construct

    2) Product Images from "The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness"

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness

    Journal: Viruses

    doi: 10.3390/v10070343

    MotB copurifies with H-NS and StpA. An SDS-PAGE gel, stained with Coomassie, is shown for the following samples obtained from BL21(DE3)/pLysE containing either the vector pTXB1 (lanes 3–10) or pTXB1-MotB (lanes 11–18): lysate applied to chitin resin (lanes 3 and 11); column flow through (lanes 4 and 12); resin sample after flow through (lanes 5 and 13); first and second column wash with buffer containing 500 mM NaCl (lanes 6, 7 and 14, 15); resin sample after second 500 mM salt wash (lanes 8 and 16); column wash with buffer containing 1 M NaCl (lanes 9 and 17); final resin sample after 1 M salt wash (lanes 10 and 18). Lane 1 corresponds to SeeBlue Plus2 protein standard; corresponding molecular weights are shown on the left. Bands corresponding to MotB-Intein/CBD and Intein/CBD are indicated. Species indicated with arrows were identified by mass spectrometry. The ~36 kDa protein labeled as “degraded construct” was identified as partially degraded MotB-Intein/CBD. The ~16 kDa protein was identified as H-NS and StpA.
    Figure Legend Snippet: MotB copurifies with H-NS and StpA. An SDS-PAGE gel, stained with Coomassie, is shown for the following samples obtained from BL21(DE3)/pLysE containing either the vector pTXB1 (lanes 3–10) or pTXB1-MotB (lanes 11–18): lysate applied to chitin resin (lanes 3 and 11); column flow through (lanes 4 and 12); resin sample after flow through (lanes 5 and 13); first and second column wash with buffer containing 500 mM NaCl (lanes 6, 7 and 14, 15); resin sample after second 500 mM salt wash (lanes 8 and 16); column wash with buffer containing 1 M NaCl (lanes 9 and 17); final resin sample after 1 M salt wash (lanes 10 and 18). Lane 1 corresponds to SeeBlue Plus2 protein standard; corresponding molecular weights are shown on the left. Bands corresponding to MotB-Intein/CBD and Intein/CBD are indicated. Species indicated with arrows were identified by mass spectrometry. The ~36 kDa protein labeled as “degraded construct” was identified as partially degraded MotB-Intein/CBD. The ~16 kDa protein was identified as H-NS and StpA.

    Techniques Used: SDS Page, Staining, Plasmid Preparation, Mass Spectrometry, Labeling, Construct

    3) Product Images from "The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness"

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness

    Journal: Viruses

    doi: 10.3390/v10070343

    MotB copurifies with H-NS and StpA. An SDS-PAGE gel, stained with Coomassie, is shown for the following samples obtained from BL21(DE3)/pLysE containing either the vector pTXB1 (lanes 3–10) or pTXB1-MotB (lanes 11–18): lysate applied to chitin resin (lanes 3 and 11); column flow through (lanes 4 and 12); resin sample after flow through (lanes 5 and 13); first and second column wash with buffer containing 500 mM NaCl (lanes 6, 7 and 14, 15); resin sample after second 500 mM salt wash (lanes 8 and 16); column wash with buffer containing 1 M NaCl (lanes 9 and 17); final resin sample after 1 M salt wash (lanes 10 and 18). Lane 1 corresponds to SeeBlue Plus2 protein standard; corresponding molecular weights are shown on the left. Bands corresponding to MotB-Intein/CBD and Intein/CBD are indicated. Species indicated with arrows were identified by mass spectrometry. The ~36 kDa protein labeled as “degraded construct” was identified as partially degraded MotB-Intein/CBD. The ~16 kDa protein was identified as H-NS and StpA.
    Figure Legend Snippet: MotB copurifies with H-NS and StpA. An SDS-PAGE gel, stained with Coomassie, is shown for the following samples obtained from BL21(DE3)/pLysE containing either the vector pTXB1 (lanes 3–10) or pTXB1-MotB (lanes 11–18): lysate applied to chitin resin (lanes 3 and 11); column flow through (lanes 4 and 12); resin sample after flow through (lanes 5 and 13); first and second column wash with buffer containing 500 mM NaCl (lanes 6, 7 and 14, 15); resin sample after second 500 mM salt wash (lanes 8 and 16); column wash with buffer containing 1 M NaCl (lanes 9 and 17); final resin sample after 1 M salt wash (lanes 10 and 18). Lane 1 corresponds to SeeBlue Plus2 protein standard; corresponding molecular weights are shown on the left. Bands corresponding to MotB-Intein/CBD and Intein/CBD are indicated. Species indicated with arrows were identified by mass spectrometry. The ~36 kDa protein labeled as “degraded construct” was identified as partially degraded MotB-Intein/CBD. The ~16 kDa protein was identified as H-NS and StpA.

    Techniques Used: SDS Page, Staining, Plasmid Preparation, Flow Cytometry, Mass Spectrometry, Labeling, Construct

    Related Articles

    Plasmid Preparation:

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: Site directed mutagenesis was performed using a Quickchange II XL Site-Directed Mutagenesis Kit (Strategene). .. 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. .. The ligation reaction was transformed into XL10-Gold Ultracompetent cells (Stratagene) and resulting colonies were screened on (LB) plates containing amp.

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: Amp-resistant colonies were then picked and cultured. .. Plasmid DNA was isolated by miniprep and then sequenced (using T7short and T7 terminator primers) to confirm the mutation, and to confirm that D23N-Aβ1-29 was correctly aligned into the pTXB1 vector. .. Vector DNA was transformed into BL21DE3 or ER2566 cells and plated onto LB-amp plates.

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: .. DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: The ligation reaction was transformed into ER2566 cells and resulting colonies were screened on Luria broth (LB) plates containing ampicillin (amp) and using restriction digest. .. Sequencing verified that Aβ1-29 was correctly aligned into the pTXB1 vector. .. For production of Aβ peptides bearing a point mutation associated with Familial Alzheimer’s Disease, we performed site directed mutagenesis to obtain a construct with the Iowa mutation, D23N.

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: The PCR cycle included 95 °C for 20 minutes, 30 cycles of 95 °C for 30 seconds, 46 °C for 30 seconds and 72 °C for 1 minute and finally 72 °C for 5 minutes. .. PCR reaction utilized TAq polymerase (Roche). pTXB1 vector (New England Biolabs) and PCR product were digested with Nde1 and Sap1 (New England Biolabs) and dephosphorylated using calf intestinal alkaline phosphatase (Invitrogen). .. The vector and insert were ligated using T4 DNA ligase (Fermentas).

    Article Title: Facile Method for the Site-Specific, Covalent Attachment of full-length IgG onto Nanoparticles
    Article Snippet: .. The resulting Protein Z sequence was gel purified and directly ligated with gel-purified Nde I- Sap I double digested pTXB1 vector (New England Biolabs, Inc) via the CloneEZ kit (Genscript). .. Insertion of the Protein Z sequence was verified by DNA sequencing using the T7 promoter as the sequencing primer.

    Polymerase Chain Reaction:

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: Site directed mutagenesis was performed using a Quickchange II XL Site-Directed Mutagenesis Kit (Strategene). .. 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. .. The ligation reaction was transformed into XL10-Gold Ultracompetent cells (Stratagene) and resulting colonies were screened on (LB) plates containing amp.

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: The PCR cycle included 95 °C for 20 minutes, 30 cycles of 95 °C for 30 seconds, 46 °C for 30 seconds and 72 °C for 1 minute and finally 72 °C for 5 minutes. .. PCR reaction utilized TAq polymerase (Roche). pTXB1 vector (New England Biolabs) and PCR product were digested with Nde1 and Sap1 (New England Biolabs) and dephosphorylated using calf intestinal alkaline phosphatase (Invitrogen). .. The vector and insert were ligated using T4 DNA ligase (Fermentas).

    Isolation:

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: Amp-resistant colonies were then picked and cultured. .. Plasmid DNA was isolated by miniprep and then sequenced (using T7short and T7 terminator primers) to confirm the mutation, and to confirm that D23N-Aβ1-29 was correctly aligned into the pTXB1 vector. .. Vector DNA was transformed into BL21DE3 or ER2566 cells and plated onto LB-amp plates.

    Mutagenesis:

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: Amp-resistant colonies were then picked and cultured. .. Plasmid DNA was isolated by miniprep and then sequenced (using T7short and T7 terminator primers) to confirm the mutation, and to confirm that D23N-Aβ1-29 was correctly aligned into the pTXB1 vector. .. Vector DNA was transformed into BL21DE3 or ER2566 cells and plated onto LB-amp plates.

    Clone Assay:

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: .. DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness
    Article Snippet: DNA pTE103-motBam was prepared by cloning the 489 base pair (bp) sequence preceding and following the codon for motB residue S12, mutated from TCT to TAG, between the BamHI and SalI sites. pNW129-MotB was constructed by cloning motB , whose sequence was optimized for codon usage, between KpnI and SalI sites of the kanr , pACYC-derived vector pNW129 [ ]. .. In this arrangement, motB is appropriately downstream of a ribosome-binding site and is under the control of the arabinose inducible promoter PBAD . pNW129-MotB-His was constructed as described for pNW129-MotB except the native stop codon was omitted so that the C-terminal His6 -tag was transcribed. pTXB1-MotB was obtained by cloning the native sequence minus its stop codon between the NdeI and SapI sites of pTXB1 (New England BioLabs, Ipswich, MA, USA). .. In this arrangement, motB is appropriately downstream of a ribosome-binding site and is under the control of the arabinose inducible promoter PBAD . pNW129-MotB-His was constructed as described for pNW129-MotB except the native stop codon was omitted so that the C-terminal His6 -tag was transcribed. pTXB1-MotB was obtained by cloning the native sequence minus its stop codon between the NdeI and SapI sites of pTXB1 (New England BioLabs, Ipswich, MA, USA).

    Ligation:

    Article Title: Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser422 containing epitope on pathological tau
    Article Snippet: .. DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs). .. After cloning, the plasmid was amplified in E. coli NEB5α cells (New England Biolabs).

    Construct:

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness
    Article Snippet: DNA pTE103-motBam was prepared by cloning the 489 base pair (bp) sequence preceding and following the codon for motB residue S12, mutated from TCT to TAG, between the BamHI and SalI sites. pNW129-MotB was constructed by cloning motB , whose sequence was optimized for codon usage, between KpnI and SalI sites of the kanr , pACYC-derived vector pNW129 [ ]. .. In this arrangement, motB is appropriately downstream of a ribosome-binding site and is under the control of the arabinose inducible promoter PBAD . pNW129-MotB-His was constructed as described for pNW129-MotB except the native stop codon was omitted so that the C-terminal His6 -tag was transcribed. pTXB1-MotB was obtained by cloning the native sequence minus its stop codon between the NdeI and SapI sites of pTXB1 (New England BioLabs, Ipswich, MA, USA). .. In this arrangement, motB is appropriately downstream of a ribosome-binding site and is under the control of the arabinose inducible promoter PBAD . pNW129-MotB-His was constructed as described for pNW129-MotB except the native stop codon was omitted so that the C-terminal His6 -tag was transcribed. pTXB1-MotB was obtained by cloning the native sequence minus its stop codon between the NdeI and SapI sites of pTXB1 (New England BioLabs, Ipswich, MA, USA).

    Sequencing:

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness
    Article Snippet: DNA pTE103-motBam was prepared by cloning the 489 base pair (bp) sequence preceding and following the codon for motB residue S12, mutated from TCT to TAG, between the BamHI and SalI sites. pNW129-MotB was constructed by cloning motB , whose sequence was optimized for codon usage, between KpnI and SalI sites of the kanr , pACYC-derived vector pNW129 [ ]. .. In this arrangement, motB is appropriately downstream of a ribosome-binding site and is under the control of the arabinose inducible promoter PBAD . pNW129-MotB-His was constructed as described for pNW129-MotB except the native stop codon was omitted so that the C-terminal His6 -tag was transcribed. pTXB1-MotB was obtained by cloning the native sequence minus its stop codon between the NdeI and SapI sites of pTXB1 (New England BioLabs, Ipswich, MA, USA). .. In this arrangement, motB is appropriately downstream of a ribosome-binding site and is under the control of the arabinose inducible promoter PBAD . pNW129-MotB-His was constructed as described for pNW129-MotB except the native stop codon was omitted so that the C-terminal His6 -tag was transcribed. pTXB1-MotB was obtained by cloning the native sequence minus its stop codon between the NdeI and SapI sites of pTXB1 (New England BioLabs, Ipswich, MA, USA).

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides
    Article Snippet: The ligation reaction was transformed into ER2566 cells and resulting colonies were screened on Luria broth (LB) plates containing ampicillin (amp) and using restriction digest. .. Sequencing verified that Aβ1-29 was correctly aligned into the pTXB1 vector. .. For production of Aβ peptides bearing a point mutation associated with Familial Alzheimer’s Disease, we performed site directed mutagenesis to obtain a construct with the Iowa mutation, D23N.

    Article Title: Facile Method for the Site-Specific, Covalent Attachment of full-length IgG onto Nanoparticles
    Article Snippet: .. The resulting Protein Z sequence was gel purified and directly ligated with gel-purified Nde I- Sap I double digested pTXB1 vector (New England Biolabs, Inc) via the CloneEZ kit (Genscript). .. Insertion of the Protein Z sequence was verified by DNA sequencing using the T7 promoter as the sequencing primer.

    Purification:

    Article Title: Facile Method for the Site-Specific, Covalent Attachment of full-length IgG onto Nanoparticles
    Article Snippet: .. The resulting Protein Z sequence was gel purified and directly ligated with gel-purified Nde I- Sap I double digested pTXB1 vector (New England Biolabs, Inc) via the CloneEZ kit (Genscript). .. Insertion of the Protein Z sequence was verified by DNA sequencing using the T7 promoter as the sequencing primer.

    other:

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness
    Article Snippet: BL21(DE3)/pLysE containing pTXB1-MotB or pTXB1 was grown in LB containing 25 μg/mL chloramphenicol and 100 μg/mL carbenicillin at 25 °C.

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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: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ptxb1 vector/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ptxb1 vector - by Bioz Stars, 2021-04
    97/100 stars
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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: Sequencing verified that Aβ1-29 was correctly aligned into the pTXB1 vector.

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

    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

    Journal: Acta Neuropathologica Communications

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

    doi: 10.1186/s40478-018-0562-9

    Figure Lengend 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

    Article Snippet: DNA inserts for tau fragments with suitable overlaps to the pTXB1 vector (New England Biolabs) were obtained commercially (IDT) and cloned into the expressed protein ligation vector using the IMPACT kit and associated protocols (New England Biolabs).

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