ptxb1  (New England Biolabs)


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    Structured Review

    New England Biolabs ptxb1
    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 <t>pTXB1</t> (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.
    Ptxb1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ptxb1/product/New England Biolabs
    Average 95 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    ptxb1 - by Bioz Stars, 2022-09
    95/100 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 "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

    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

    4) 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

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    New England Biolabs ptxb1 vector dna
    The experiment concept and validation of the recombinant protein pA-M.EcoGII in BIND MODIFY. (A)The experiment concept of the BIND MODIFY. The cells were lightly fixed and permeabilized for the antibody and enzyme passage. The recombinant protein pA-M.EcoGII was located to desired sites under antibody guidance. Then the M.EcoGII activity was locally activated to modify the nearby regions to label the genomic DNA with targeted binding proteins. (B) The upper panel showed the plasmid map of the pA-M.EcoGII. The fused pA-M.EcoGII was cloned into <t>pTXB1</t> plasmid and purified with compatible IMPACT protein purification system. The lower panel showed the expressed fusion protein structure: Protein A-linker-M.EcoGII-intein-CBD. (C) The Coomassie blue gel stain showed the purity of the purified protein A-M.EcoGII. (D) Methylation of linear lambda DNA by pA-M.EcoGII activates m6A-site dependent DpnI restriction endonuclease digestion. The PCR amplified unmethylated lambda DNA was treated with commercial M.EcoGII, no enzyme, and pA-M.EcoGII. The G A TC m6A methylation dependent restriction endonuclease DpnI digestion showed the comparable methyltransferase activity of the commercial M.EcoGII and our recombinant proteins. DNA marker: 100bp ladder, 100-1510bp(left); 1kb ladder, 250-10,000bp(right). (E) Methylation of linear dsDNA by pA-M.EcoGII inhibits multiple site-specific methylation sensitive restriction endonucleases. The unmethylated DNA template was a 7kb linear dsDNA, which was PCR amplified from pTXB1 plasmid. The DNA template was treated with commercial M.EcoGII, pA-M.EcoGII and no enzyme. These treated DNA templates were each incubated with four restriction endonucleases (BamHI, EcoRV, PciI, PvuII). The BamHI is the m6A methylation insensitive enzyme, and the EcoRV, PciI, PvuII are the m6A methylation sensitive enzyme, with which the digestion could be blocked by corresponding m6A site. Our pA-M.EcoGII recombinant protein showed digestion inhibition on EcoRV, PciI, PvuII digested samples, better than commercial M.EcoGII, as compared to untreated DNA template. DNA marker, 1kb ladder, 250-10,000bp. (F) The antibody affinity assay showed the recombinant pA-M.EcoGII had the affinity to the secondary antibody in two different dilutions (1/120,1/480, 10mg/ml). (G) The experiment outlines of BIND MODIFY. After light fixation and permeabilization, the cells were tethered to Concanavalin A magnetic beads for the purification in the next steps. Then the cells were incubated with antibody and pA-M.EcoGII with minimal washes. The addition of S-adenosylmethionine to initialize the methylation reaction. The DNA was extracted to prepare the library for ONT nanopore sequencing. After sequencing, the data was processed as genome alignment and m6A base calling.
    Ptxb1 Vector Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ptxb1 vector dna - by Bioz Stars, 2022-09
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    The experiment concept and validation of the recombinant protein pA-M.EcoGII in BIND MODIFY. (A)The experiment concept of the BIND MODIFY. The cells were lightly fixed and permeabilized for the antibody and enzyme passage. The recombinant protein pA-M.EcoGII was located to desired sites under antibody guidance. Then the M.EcoGII activity was locally activated to modify the nearby regions to label the genomic DNA with targeted binding proteins. (B) The upper panel showed the plasmid map of the pA-M.EcoGII. The fused pA-M.EcoGII was cloned into pTXB1 plasmid and purified with compatible IMPACT protein purification system. The lower panel showed the expressed fusion protein structure: Protein A-linker-M.EcoGII-intein-CBD. (C) The Coomassie blue gel stain showed the purity of the purified protein A-M.EcoGII. (D) Methylation of linear lambda DNA by pA-M.EcoGII activates m6A-site dependent DpnI restriction endonuclease digestion. The PCR amplified unmethylated lambda DNA was treated with commercial M.EcoGII, no enzyme, and pA-M.EcoGII. The G A TC m6A methylation dependent restriction endonuclease DpnI digestion showed the comparable methyltransferase activity of the commercial M.EcoGII and our recombinant proteins. DNA marker: 100bp ladder, 100-1510bp(left); 1kb ladder, 250-10,000bp(right). (E) Methylation of linear dsDNA by pA-M.EcoGII inhibits multiple site-specific methylation sensitive restriction endonucleases. The unmethylated DNA template was a 7kb linear dsDNA, which was PCR amplified from pTXB1 plasmid. The DNA template was treated with commercial M.EcoGII, pA-M.EcoGII and no enzyme. These treated DNA templates were each incubated with four restriction endonucleases (BamHI, EcoRV, PciI, PvuII). The BamHI is the m6A methylation insensitive enzyme, and the EcoRV, PciI, PvuII are the m6A methylation sensitive enzyme, with which the digestion could be blocked by corresponding m6A site. Our pA-M.EcoGII recombinant protein showed digestion inhibition on EcoRV, PciI, PvuII digested samples, better than commercial M.EcoGII, as compared to untreated DNA template. DNA marker, 1kb ladder, 250-10,000bp. (F) The antibody affinity assay showed the recombinant pA-M.EcoGII had the affinity to the secondary antibody in two different dilutions (1/120,1/480, 10mg/ml). (G) The experiment outlines of BIND MODIFY. After light fixation and permeabilization, the cells were tethered to Concanavalin A magnetic beads for the purification in the next steps. Then the cells were incubated with antibody and pA-M.EcoGII with minimal washes. The addition of S-adenosylmethionine to initialize the methylation reaction. The DNA was extracted to prepare the library for ONT nanopore sequencing. After sequencing, the data was processed as genome alignment and m6A base calling.

    Journal: bioRxiv

    Article Title: Long-range single-molecule mapping of chromatin modification in eukaryotes

    doi: 10.1101/2021.07.08.451578

    Figure Lengend Snippet: The experiment concept and validation of the recombinant protein pA-M.EcoGII in BIND MODIFY. (A)The experiment concept of the BIND MODIFY. The cells were lightly fixed and permeabilized for the antibody and enzyme passage. The recombinant protein pA-M.EcoGII was located to desired sites under antibody guidance. Then the M.EcoGII activity was locally activated to modify the nearby regions to label the genomic DNA with targeted binding proteins. (B) The upper panel showed the plasmid map of the pA-M.EcoGII. The fused pA-M.EcoGII was cloned into pTXB1 plasmid and purified with compatible IMPACT protein purification system. The lower panel showed the expressed fusion protein structure: Protein A-linker-M.EcoGII-intein-CBD. (C) The Coomassie blue gel stain showed the purity of the purified protein A-M.EcoGII. (D) Methylation of linear lambda DNA by pA-M.EcoGII activates m6A-site dependent DpnI restriction endonuclease digestion. The PCR amplified unmethylated lambda DNA was treated with commercial M.EcoGII, no enzyme, and pA-M.EcoGII. The G A TC m6A methylation dependent restriction endonuclease DpnI digestion showed the comparable methyltransferase activity of the commercial M.EcoGII and our recombinant proteins. DNA marker: 100bp ladder, 100-1510bp(left); 1kb ladder, 250-10,000bp(right). (E) Methylation of linear dsDNA by pA-M.EcoGII inhibits multiple site-specific methylation sensitive restriction endonucleases. The unmethylated DNA template was a 7kb linear dsDNA, which was PCR amplified from pTXB1 plasmid. The DNA template was treated with commercial M.EcoGII, pA-M.EcoGII and no enzyme. These treated DNA templates were each incubated with four restriction endonucleases (BamHI, EcoRV, PciI, PvuII). The BamHI is the m6A methylation insensitive enzyme, and the EcoRV, PciI, PvuII are the m6A methylation sensitive enzyme, with which the digestion could be blocked by corresponding m6A site. Our pA-M.EcoGII recombinant protein showed digestion inhibition on EcoRV, PciI, PvuII digested samples, better than commercial M.EcoGII, as compared to untreated DNA template. DNA marker, 1kb ladder, 250-10,000bp. (F) The antibody affinity assay showed the recombinant pA-M.EcoGII had the affinity to the secondary antibody in two different dilutions (1/120,1/480, 10mg/ml). (G) The experiment outlines of BIND MODIFY. After light fixation and permeabilization, the cells were tethered to Concanavalin A magnetic beads for the purification in the next steps. Then the cells were incubated with antibody and pA-M.EcoGII with minimal washes. The addition of S-adenosylmethionine to initialize the methylation reaction. The DNA was extracted to prepare the library for ONT nanopore sequencing. After sequencing, the data was processed as genome alignment and m6A base calling.

    Article Snippet: Recombinant protein preparation of protein A-M.EcoGIIpTXB1 vector (NEB, N6707S) was used as the protein expression backbone.

    Techniques: Recombinant, Activity Assay, Binding Assay, Plasmid Preparation, Clone Assay, Purification, Protein Purification, Staining, Methylation, Lambda DNA Preparation, Polymerase Chain Reaction, Amplification, Marker, Incubation, Inhibition, Magnetic Beads, Nanopore Sequencing, Sequencing

    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