tris hcl  (New England Biolabs)


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    Structured Review

    New England Biolabs tris hcl
    PAn from H17N10 has endonuclease activity. (A) Single-stranded DNA plasmid M13mp18 (50 ng/μl) was incubated with 20 μM wild-type H17N10 PAn for 1 h at 37°C in a buffer consisting of 20 mM <t>Tris</t> (pH 8) and 50 mM <t>NaCl</t> in the absence
    Tris Hcl, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity"

    Article Title: The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity

    Journal: Journal of Virology

    doi: 10.1128/JVI.03270-13

    PAn from H17N10 has endonuclease activity. (A) Single-stranded DNA plasmid M13mp18 (50 ng/μl) was incubated with 20 μM wild-type H17N10 PAn for 1 h at 37°C in a buffer consisting of 20 mM Tris (pH 8) and 50 mM NaCl in the absence
    Figure Legend Snippet: PAn from H17N10 has endonuclease activity. (A) Single-stranded DNA plasmid M13mp18 (50 ng/μl) was incubated with 20 μM wild-type H17N10 PAn for 1 h at 37°C in a buffer consisting of 20 mM Tris (pH 8) and 50 mM NaCl in the absence

    Techniques Used: Activity Assay, Plasmid Preparation, Incubation

    2) Product Images from "The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms"

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002534

    Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).
    Figure Legend Snippet: Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).

    Techniques Used: Incubation, Labeling, Sequencing

    3) Product Images from "Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6"

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    Journal: PLoS Computational Biology

    doi: 10.1371/journal.pcbi.1004690

    Coq6p biochemical characterization. A) SDS PAGE (4–12% Bis-Tris, MOPS). Lane 1, Molecular Weights (kDa); lane 2, BL21 DE3 cells transformed with pMALc2X-Coq6 before IPTG induction; lane 3, cells after 20 hr-IPTG induction at 18°C; lane 4, Coq6p-MBP (96.3 kDa) after a two-step purification (MBP trap, Superdex200 26/60). B) UV-visible spectrum of purified Coq6p-MBP, 1.20 mg/ml.
    Figure Legend Snippet: Coq6p biochemical characterization. A) SDS PAGE (4–12% Bis-Tris, MOPS). Lane 1, Molecular Weights (kDa); lane 2, BL21 DE3 cells transformed with pMALc2X-Coq6 before IPTG induction; lane 3, cells after 20 hr-IPTG induction at 18°C; lane 4, Coq6p-MBP (96.3 kDa) after a two-step purification (MBP trap, Superdex200 26/60). B) UV-visible spectrum of purified Coq6p-MBP, 1.20 mg/ml.

    Techniques Used: SDS Page, Transformation Assay, Purification

    4) Product Images from "Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase"

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl060

    Size-exclusion chromatographic analysis of wild-type and S326C OGG1. ( A ) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min ( B ). Identical runs were performed with 100 µg polymorphic S326C OGG1 ( C ) or 100 µg of both wild-type and S326C OGG1 together ( D ).
    Figure Legend Snippet: Size-exclusion chromatographic analysis of wild-type and S326C OGG1. ( A ) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min ( B ). Identical runs were performed with 100 µg polymorphic S326C OGG1 ( C ) or 100 µg of both wild-type and S326C OGG1 together ( D ).

    Techniques Used: Purification, Flow Cytometry

    5) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    6) Product Images from "The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms"

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002534

    Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).
    Figure Legend Snippet: Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).

    Techniques Used: Incubation, Labeling, Sequencing

    7) Product Images from "The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms"

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002534

    Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).
    Figure Legend Snippet: Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).

    Techniques Used: Incubation, Labeling, Sequencing

    8) Product Images from "Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions"

    Article Title: Canonical nucleosides can be utilized by T4 DNA ligase as universal template bases at ligation junctions

    Journal: Nucleic Acids Research

    doi:

    Time-dependent ligation reactions. The optimum reaction conditions were used, which are 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , 10 µM ATP, 20% DMSO, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol C4 template and 3 U T4 DNA ligase at 22°C. The graph shows the plots and fitted curves of each of the four ligation reactions analyzed (see inset for oligonucleotide sequences). The observed rate constants ( k obs ) for each reaction are given on the graph. The data points are the average of two independent assays.
    Figure Legend Snippet: Time-dependent ligation reactions. The optimum reaction conditions were used, which are 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , 10 µM ATP, 20% DMSO, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol C4 template and 3 U T4 DNA ligase at 22°C. The graph shows the plots and fitted curves of each of the four ligation reactions analyzed (see inset for oligonucleotide sequences). The observed rate constants ( k obs ) for each reaction are given on the graph. The data points are the average of two independent assays.

    Techniques Used: Ligation

    Optimization of ligation reactions. ( A and B ) ATP and MgCl 2 concentration-dependent ligations using (c + p c)/T2. The MgCl 2 concentration was first optimized under standard reaction conditions of 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 1 mM ATP, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol template and 3 U T4 DNA ligase at 30°C for 18 h. An optimum concentration of 3 mM MgCl 2 was found, which was then used in the subsequent ATP-dependent assay. ( C ) DMSO-dependent ligation reaction using (c + p c)/C2 in 3 mM MgCl 2 and 10 µM ATP. ( D ) Time-dependent ligation reaction using (c + p c)/T2 in 3 mM MgCl 2 , 10 µM ATP and 20% DMSO. In each case, product is represented by squares and intermediate by circles.
    Figure Legend Snippet: Optimization of ligation reactions. ( A and B ) ATP and MgCl 2 concentration-dependent ligations using (c + p c)/T2. The MgCl 2 concentration was first optimized under standard reaction conditions of 30 mM Tris–HCl (pH 7.5), 10 mM DTT, 1 mM ATP, 2.5 pmol acceptor molecule, 70 fmol donor molecule, 3.125 pmol template and 3 U T4 DNA ligase at 30°C for 18 h. An optimum concentration of 3 mM MgCl 2 was found, which was then used in the subsequent ATP-dependent assay. ( C ) DMSO-dependent ligation reaction using (c + p c)/C2 in 3 mM MgCl 2 and 10 µM ATP. ( D ) Time-dependent ligation reaction using (c + p c)/T2 in 3 mM MgCl 2 , 10 µM ATP and 20% DMSO. In each case, product is represented by squares and intermediate by circles.

    Techniques Used: Ligation, Concentration Assay

    9) Product Images from "High-Resolution X-Ray Structure of the Trimeric Scar/WAVE-Complex Precursor Brk1"

    Article Title: High-Resolution X-Ray Structure of the Trimeric Scar/WAVE-Complex Precursor Brk1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021327

    DdBrk1 chains swap within DdBrk1-containing complexes. MBP-DdBrk1 homotrimers and Scar(1-225)/AbiA(1-149)/DdBrk1-complexes were incubated with DdBrk1_488 and subjected to gelfiltration. (A) The elution of 200 nM DdBrk1_488 alone and 100 nM DdBrk1_488 after incubation with other DdBrk1 containing complexes was monitored at 488 nm. 100 nM DdBrk1_488 showed an increased r H in the presence of 1.4 µM MBP-DdBrk1- and Scar(1–225)/AbiA(1–149)/DdBrk1-complexes, confirming an exchange of subunits within these complexes. The unlabeled complexes were undetectable in the absence of DdBrk1_488 at 488 nm absorption. (B) Simultaneously recorded elution profiles at 280 nm showed slightly decreased hydrodynamic radii of the MBP-DdBrk1 fusion construct after incubation with DdBrk1_488, indicating different stoichiometries of trimeric MBP-DdBrk1/DdBrk1_488-complexes. The inset shows a 16% Tris-Tricine SDS-PAGE stained with Coomassie blue of copurified Scar(1–225)/AbiA(1–149)/DdBrk1 used in these experiments. For reasons of clarity, the different complexes are schematically shown in addition to the elution profiles. (C) Sedimentation velocity experiments of DdBrk1_488 showed dissociation of the homotrimer in the lower nanomolar range. The decreasing s-value of the faster sedimenting boundary at decreasing protein concentrations indicates a fast dissociation of the homotrimer when compared to the time scale of the experiment. The inset shows a 16% Tris-Tricine SDS-PAGE of DdBrk1_488 visualized by Coomassie-blue stain and ultraviolet illumination.
    Figure Legend Snippet: DdBrk1 chains swap within DdBrk1-containing complexes. MBP-DdBrk1 homotrimers and Scar(1-225)/AbiA(1-149)/DdBrk1-complexes were incubated with DdBrk1_488 and subjected to gelfiltration. (A) The elution of 200 nM DdBrk1_488 alone and 100 nM DdBrk1_488 after incubation with other DdBrk1 containing complexes was monitored at 488 nm. 100 nM DdBrk1_488 showed an increased r H in the presence of 1.4 µM MBP-DdBrk1- and Scar(1–225)/AbiA(1–149)/DdBrk1-complexes, confirming an exchange of subunits within these complexes. The unlabeled complexes were undetectable in the absence of DdBrk1_488 at 488 nm absorption. (B) Simultaneously recorded elution profiles at 280 nm showed slightly decreased hydrodynamic radii of the MBP-DdBrk1 fusion construct after incubation with DdBrk1_488, indicating different stoichiometries of trimeric MBP-DdBrk1/DdBrk1_488-complexes. The inset shows a 16% Tris-Tricine SDS-PAGE stained with Coomassie blue of copurified Scar(1–225)/AbiA(1–149)/DdBrk1 used in these experiments. For reasons of clarity, the different complexes are schematically shown in addition to the elution profiles. (C) Sedimentation velocity experiments of DdBrk1_488 showed dissociation of the homotrimer in the lower nanomolar range. The decreasing s-value of the faster sedimenting boundary at decreasing protein concentrations indicates a fast dissociation of the homotrimer when compared to the time scale of the experiment. The inset shows a 16% Tris-Tricine SDS-PAGE of DdBrk1_488 visualized by Coomassie-blue stain and ultraviolet illumination.

    Techniques Used: Incubation, Construct, SDS Page, Staining, Sedimentation

    DdBrk1 and PirA remain stable in the absence of Scar. (A) DdBrk1 is evolutionary conserved. Multiple sequence alignment of Brk1 from different species using the MUSCA algorithm [56] . Black letters show non-similar residues; letters in identical color display amino acids with similar hydropathy; stars on top depict blocks of conserved amino acids and open circles below indicate identical residues. At: Arabidopsis thaliana (NP_179849); Dd: Dictyostelium discoideum (XP_641829); Dm: Drosophila melanogaster (NP_726400); Hs: Homo sapiens (AAF28978). (B) The specificity of anti-DdBrk1 polyclonal antibodies was assessed by Western blotting. (Left) MBP and MBP-DdBrk1 were separated by a 10% SDS-PAGE and visualized by Coomassie-blue stain. (Right) Immunoblot analysis of the same proteins after transfer onto a PVDF membrane and detection with anti-DdBrk1 antibodies. The antibodies specifically detected the DdBrk1 containing sample, but did not bind to the MBP moiety, which served as a negative control. (C) DdBrk1 and PirA remain stable in Scar-null cells. Total cellular proteins corresponding to 2×10 5 wild-type or Scar-null cells were separated by SDS-PAGE using 16% Tris-Tricine or 10% Tris-Glycine gels, transferred to a PVDF membrane and labelled by anti-DdBrk1 antibodies. The same samples were also probed with anti-PirA antibodies. The Western blot with anti-actin antibody mAb 224–236–1 [45] shows equal sample loading. Densitometric analysis of stained bands revealed that DdBrk1 was moderately reduced while the PirA level was unchanged in the absence of Scar.
    Figure Legend Snippet: DdBrk1 and PirA remain stable in the absence of Scar. (A) DdBrk1 is evolutionary conserved. Multiple sequence alignment of Brk1 from different species using the MUSCA algorithm [56] . Black letters show non-similar residues; letters in identical color display amino acids with similar hydropathy; stars on top depict blocks of conserved amino acids and open circles below indicate identical residues. At: Arabidopsis thaliana (NP_179849); Dd: Dictyostelium discoideum (XP_641829); Dm: Drosophila melanogaster (NP_726400); Hs: Homo sapiens (AAF28978). (B) The specificity of anti-DdBrk1 polyclonal antibodies was assessed by Western blotting. (Left) MBP and MBP-DdBrk1 were separated by a 10% SDS-PAGE and visualized by Coomassie-blue stain. (Right) Immunoblot analysis of the same proteins after transfer onto a PVDF membrane and detection with anti-DdBrk1 antibodies. The antibodies specifically detected the DdBrk1 containing sample, but did not bind to the MBP moiety, which served as a negative control. (C) DdBrk1 and PirA remain stable in Scar-null cells. Total cellular proteins corresponding to 2×10 5 wild-type or Scar-null cells were separated by SDS-PAGE using 16% Tris-Tricine or 10% Tris-Glycine gels, transferred to a PVDF membrane and labelled by anti-DdBrk1 antibodies. The same samples were also probed with anti-PirA antibodies. The Western blot with anti-actin antibody mAb 224–236–1 [45] shows equal sample loading. Densitometric analysis of stained bands revealed that DdBrk1 was moderately reduced while the PirA level was unchanged in the absence of Scar.

    Techniques Used: Sequencing, Western Blot, SDS Page, Staining, Negative Control

    DdBrk1 forms stable trimers in solution. Analytical ultracentrifugation experiments of 43 µM DdBrk1 in PBS at a detection wavelength of 280 nm. (A) Sedimentation equilibrium gradients were measured at rotor speeds of 18,000 rpm and 26,000 rpm at 10°C. Global fitting of the data with a model of a single species using the program BPCfit [46] yielded a molar mass of 24.7 (±2) kg/mol (solid lines) indicating that the protein forms trimers in solution. (B) Sedimentation coefficient distribution as obtained from a sedimentation velocity experiment at 20°C using the program SEDFIT [48] . DdBrk1 sediments as a single species with a sedimentation coefficient s 20,W = 2.1 S. Experiments performed in a concentration rage of 15 µM to 340 µM DdBrk1 gave no indication of aggregation or dissociation of the DdBrk1 trimers, as indicated by a slight decrease of the sedimentation coefficient with increasing protein concentration (data not shown). The inset shows a Coomassie stained 16%-Tris/Tricin SDS-PAGE of the DdBrk1 sample used in these experiments.
    Figure Legend Snippet: DdBrk1 forms stable trimers in solution. Analytical ultracentrifugation experiments of 43 µM DdBrk1 in PBS at a detection wavelength of 280 nm. (A) Sedimentation equilibrium gradients were measured at rotor speeds of 18,000 rpm and 26,000 rpm at 10°C. Global fitting of the data with a model of a single species using the program BPCfit [46] yielded a molar mass of 24.7 (±2) kg/mol (solid lines) indicating that the protein forms trimers in solution. (B) Sedimentation coefficient distribution as obtained from a sedimentation velocity experiment at 20°C using the program SEDFIT [48] . DdBrk1 sediments as a single species with a sedimentation coefficient s 20,W = 2.1 S. Experiments performed in a concentration rage of 15 µM to 340 µM DdBrk1 gave no indication of aggregation or dissociation of the DdBrk1 trimers, as indicated by a slight decrease of the sedimentation coefficient with increasing protein concentration (data not shown). The inset shows a Coomassie stained 16%-Tris/Tricin SDS-PAGE of the DdBrk1 sample used in these experiments.

    Techniques Used: Sedimentation, Concentration Assay, Protein Concentration, Staining, SDS Page

    Related Articles

    Mutagenesis:

    Article Title: The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity
    Article Snippet: .. Typically, in a 10-μl reaction volume of reaction buffer consisting of 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1.5 mM MnCl2 , 50 ng/μl of M13mp18 circular single-stranded DNA (ssDNA) (New England BioLabs) was used as a substrate and incubated for 60 min at 37°C in the presence or absence of either the wild-type H17N10, H1N1, H5N1, or mutant H41A PAn molecules (1.25 to 20 μM). .. The reaction products were loaded on a 0.9% agarose gel and stained with ethidium bromide.

    Incubation:

    Article Title: The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity
    Article Snippet: .. Typically, in a 10-μl reaction volume of reaction buffer consisting of 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1.5 mM MnCl2 , 50 ng/μl of M13mp18 circular single-stranded DNA (ssDNA) (New England BioLabs) was used as a substrate and incubated for 60 min at 37°C in the presence or absence of either the wild-type H17N10, H1N1, H5N1, or mutant H41A PAn molecules (1.25 to 20 μM). .. The reaction products were loaded on a 0.9% agarose gel and stained with ethidium bromide.

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. 8-oxoguanine glycosylase and AP-lyase activity assays OGG1 proteins or nuclear extracts were incubated with DNA substrates at varying concentrations in 20 µl reactions containing 20 mM Tris–HCl (pH 7.4), 100 mM NaCl and 0.15 µg/µl BSA (New England Biolabs). .. To measure glycosylase activity, reactions were terminated by adding SDS and piperidine to 5% and 200 mM, respectively.

    Activity Assay:

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase
    Article Snippet: .. 8-oxoguanine glycosylase and AP-lyase activity assays OGG1 proteins or nuclear extracts were incubated with DNA substrates at varying concentrations in 20 µl reactions containing 20 mM Tris–HCl (pH 7.4), 100 mM NaCl and 0.15 µg/µl BSA (New England Biolabs). .. To measure glycosylase activity, reactions were terminated by adding SDS and piperidine to 5% and 200 mM, respectively.

    Fast Protein Liquid Chromatography:

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms
    Article Snippet: .. The precipitated proteins were pelleted (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4°C), dissolved in 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, and run through a 10-ml amylose column (FPLC; Amylose High-Flow resin; New England BioLabs, Ipswich, MA), which was washed with 3 column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol and eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol containing 10 mM maltose. .. Fractions containing the MalE-LtrA fusion were incubated with TEV protease (80 µg/ml, 18 h, at 4°C), and imidazole was added to a final concentration of 40 mM.

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    New England Biolabs tris hcl
    PAn from H17N10 has endonuclease activity. (A) Single-stranded DNA plasmid M13mp18 (50 ng/μl) was incubated with 20 μM wild-type H17N10 PAn for 1 h at 37°C in a buffer consisting of 20 mM <t>Tris</t> (pH 8) and 50 mM <t>NaCl</t> in the absence
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    PAn from H17N10 has endonuclease activity. (A) Single-stranded DNA plasmid M13mp18 (50 ng/μl) was incubated with 20 μM wild-type H17N10 PAn for 1 h at 37°C in a buffer consisting of 20 mM Tris (pH 8) and 50 mM NaCl in the absence

    Journal: Journal of Virology

    Article Title: The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity

    doi: 10.1128/JVI.03270-13

    Figure Lengend Snippet: PAn from H17N10 has endonuclease activity. (A) Single-stranded DNA plasmid M13mp18 (50 ng/μl) was incubated with 20 μM wild-type H17N10 PAn for 1 h at 37°C in a buffer consisting of 20 mM Tris (pH 8) and 50 mM NaCl in the absence

    Article Snippet: Typically, in a 10-μl reaction volume of reaction buffer consisting of 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1.5 mM MnCl2 , 50 ng/μl of M13mp18 circular single-stranded DNA (ssDNA) (New England BioLabs) was used as a substrate and incubated for 60 min at 37°C in the presence or absence of either the wild-type H17N10, H1N1, H5N1, or mutant H41A PAn molecules (1.25 to 20 μM).

    Techniques: Activity Assay, Plasmid Preparation, Incubation

    Rate dependence of linear QPA on the length of AT-rich segment. (A) Targets and primer sequences; targets 1, 2 and 3 contain 34-nt, 20-nt and 5-nt AT-rich segments, respectively. (B) Typical dGTP-QPA (black) and dNTP-QPA profiles (colored) obtained using 0.5 μM primer, 10 nM target 2, 100 μM dGTP or 800 μM dNTP, 0.08 U/uL Bst 2.0 polymerase in 10 mM KCl, 40 mM CsCl, 2 mM MgCl 2 and 10 mM Tris-HCl, pH 8.7 at 62 °C. Dashed line corresponds to no-template control (NTC). (C) Temperature dependence of the rate of linear QPA using target 1 (blue), target 2 (red) and target 3 (black).

    Journal: Analytical methods : advancing methods and applications

    Article Title: Isothermal amplification of long DNA segments by quadruplex priming amplification

    doi: 10.1039/C8AY00843D

    Figure Lengend Snippet: Rate dependence of linear QPA on the length of AT-rich segment. (A) Targets and primer sequences; targets 1, 2 and 3 contain 34-nt, 20-nt and 5-nt AT-rich segments, respectively. (B) Typical dGTP-QPA (black) and dNTP-QPA profiles (colored) obtained using 0.5 μM primer, 10 nM target 2, 100 μM dGTP or 800 μM dNTP, 0.08 U/uL Bst 2.0 polymerase in 10 mM KCl, 40 mM CsCl, 2 mM MgCl 2 and 10 mM Tris-HCl, pH 8.7 at 62 °C. Dashed line corresponds to no-template control (NTC). (C) Temperature dependence of the rate of linear QPA using target 1 (blue), target 2 (red) and target 3 (black).

    Article Snippet: DNA polymerases ( Vent (exo-) and Bst 2.0), dNTPs and isothermal buffer (10 mM (NH4 )2 SO4 , 50 mM KCl, 2 mM MgSO4 , 0.1% Tween® 20, 20 mM Tris-HCl, pH 8.8) were purchased from New England BioLabs.

    Techniques:

    Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).

    Journal: PLoS Genetics

    Article Title: The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4-Dependent and -Independent Mechanisms

    doi: 10.1371/journal.pgen.1002534

    Figure Lengend Snippet: Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA. The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′- 32 P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) 32 P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with 32 P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed 32 P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing ( Figure 7 ) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).

    Article Snippet: The precipitated proteins were pelleted (Beckman JA-14 rotor, 14,000 rpm, 30 min, 4°C), dissolved in 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, and run through a 10-ml amylose column (FPLC; Amylose High-Flow resin; New England BioLabs, Ipswich, MA), which was washed with 3 column volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol and eluted with 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol containing 10 mM maltose.

    Techniques: Incubation, Labeling, Sequencing

    Coq6p biochemical characterization. A) SDS PAGE (4–12% Bis-Tris, MOPS). Lane 1, Molecular Weights (kDa); lane 2, BL21 DE3 cells transformed with pMALc2X-Coq6 before IPTG induction; lane 3, cells after 20 hr-IPTG induction at 18°C; lane 4, Coq6p-MBP (96.3 kDa) after a two-step purification (MBP trap, Superdex200 26/60). B) UV-visible spectrum of purified Coq6p-MBP, 1.20 mg/ml.

    Journal: PLoS Computational Biology

    Article Title: Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    doi: 10.1371/journal.pcbi.1004690

    Figure Lengend Snippet: Coq6p biochemical characterization. A) SDS PAGE (4–12% Bis-Tris, MOPS). Lane 1, Molecular Weights (kDa); lane 2, BL21 DE3 cells transformed with pMALc2X-Coq6 before IPTG induction; lane 3, cells after 20 hr-IPTG induction at 18°C; lane 4, Coq6p-MBP (96.3 kDa) after a two-step purification (MBP trap, Superdex200 26/60). B) UV-visible spectrum of purified Coq6p-MBP, 1.20 mg/ml.

    Article Snippet: Proteolytic cleavage of the MBP tag was performed by incubating the fusion protein Coq6p-MBP (1mg/ml) in 20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl2 , glycerol 5%, pH 8.0, with Factor Xa (New England Biolabs, 1/50, enzyme/substrate w/w) at 23°C overnight.

    Techniques: SDS Page, Transformation Assay, Purification