buffer 1  (New England Biolabs)


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    NEBuffer 1 1
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    NEBuffer 1 1 5 0 ml
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    New England Biolabs buffer 1
    NEBuffer 1 1
    NEBuffer 1 1 5 0 ml
    https://www.bioz.com/result/buffer 1/product/New England Biolabs
    Average 95 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    buffer 1 - by Bioz Stars, 2020-05
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    Related Articles

    Transduction:

    Article Title: Dissecting super-enhancer hierarchy based on chromatin interactions
    Article Snippet: .. The annealed oligos were cloned into pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro vector (Addgene ID: 71236) using a Golden Gate Assembly strategy including: 100 ng of circular pLV plasmid, 0.2 μM annealed oligos, 2.1 buffer (1×) (New England Biolabs), 20 U of BsmBI restriction enzyme, 0.2 mM ATP, 0.1 mg/ml BSA, and 750 U of T4 DNA ligase (New England Biolabs) with the cycling parameters of 20 cycles of 37 °C for 5 min, 20 °C for 5 min; followed by 80 °C incubation for 20 min. Then K562 cells were transduced with lentivirus to stably express dCa s9-KRAB and sgRNA. .. Briefly, sequence-specific sgRNAs for site-specific interference of genomic targets were designed following described guidelines, and sequences were selected to minimize off-target effect based on publicly available filtering tools ( http://crispr.mit.edu/ ).

    Clone Assay:

    Article Title: Dissecting super-enhancer hierarchy based on chromatin interactions
    Article Snippet: .. The annealed oligos were cloned into pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro vector (Addgene ID: 71236) using a Golden Gate Assembly strategy including: 100 ng of circular pLV plasmid, 0.2 μM annealed oligos, 2.1 buffer (1×) (New England Biolabs), 20 U of BsmBI restriction enzyme, 0.2 mM ATP, 0.1 mg/ml BSA, and 750 U of T4 DNA ligase (New England Biolabs) with the cycling parameters of 20 cycles of 37 °C for 5 min, 20 °C for 5 min; followed by 80 °C incubation for 20 min. Then K562 cells were transduced with lentivirus to stably express dCa s9-KRAB and sgRNA. .. Briefly, sequence-specific sgRNAs for site-specific interference of genomic targets were designed following described guidelines, and sequences were selected to minimize off-target effect based on publicly available filtering tools ( http://crispr.mit.edu/ ).

    Centrifugation:

    Article Title: Analysis of the Interaction between Globular Head Modules of Human C1q and Its Candidate Receptor gC1qR
    Article Snippet: .. After centrifugation (13,000 rpm for 15 min), the supernatant was collected and diluted fivefold in buffer I (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.2% Tween 20, 1 mM EDTA, and 5% v/v glycerol) and passed through an amylose resin 15 ml bed column (New England Biolabs). ..

    Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission
    Article Snippet: .. After centrifugation (13,000 × g for 15 min), the supernatant was diluted fivefold in buffer I (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.2% Tween 20, 1 mM EDTA, and 5% glycerol), passed through an amylose resin column (15 mL; New England Biolabs), and then washed with three bed volumes of buffer I followed by buffer II (50 mL of buffer I without Tween 20). .. The protein was then eluted in 1 mL fractions with 10 mM maltose in 100 mL of buffer II and frozen at −20°C after determining protein concentration and purity via Nanodrop and 10% w/v SDS-PAGE, respectively.

    Agarose Gel Electrophoresis:

    Article Title: A Simple Procedure for Creating Scalable Phenotypic Screening Assays in Human Neurons
    Article Snippet: .. Individual plasmid DNA were incubated with appropriate restriction enzymes (New England Biolabs, Ipswich, MA) and NE Buffer (cat. no. B7201, NEB) for 1 h. Purple loading dye (cat. no. B7024S, NEB) was added to each sample which were loaded carefully on MiniReady Agarose Gel prepared in 1XTBE (cat. no. 101-3004, Biorad) alongside with Quick load purple 1 kb DNA ladder as a reference standard (cat. no. N0552G, NEB). .. The results demonstrated appropriate digest products for each plasmid as well as the maxi-prep RFLP and can be found in the supplemental data (Figure ).

    Stable Transfection:

    Article Title: Dissecting super-enhancer hierarchy based on chromatin interactions
    Article Snippet: .. The annealed oligos were cloned into pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro vector (Addgene ID: 71236) using a Golden Gate Assembly strategy including: 100 ng of circular pLV plasmid, 0.2 μM annealed oligos, 2.1 buffer (1×) (New England Biolabs), 20 U of BsmBI restriction enzyme, 0.2 mM ATP, 0.1 mg/ml BSA, and 750 U of T4 DNA ligase (New England Biolabs) with the cycling parameters of 20 cycles of 37 °C for 5 min, 20 °C for 5 min; followed by 80 °C incubation for 20 min. Then K562 cells were transduced with lentivirus to stably express dCa s9-KRAB and sgRNA. .. Briefly, sequence-specific sgRNAs for site-specific interference of genomic targets were designed following described guidelines, and sequences were selected to minimize off-target effect based on publicly available filtering tools ( http://crispr.mit.edu/ ).

    Isolation:

    Article Title: The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components
    Article Snippet: .. Biochemical R.HpyAXII endonuclease activity assay One microliter of freshly prepared soluble fraction obtained from E. coli BL21(DE3) expressing R.HpyAXII was incubated for 3 h at 37°C with 500 ng E. coli ER2925 genomic DNA or pET15b plasmid DNA isolated from E. coli DH10B in presence of NEB buffer 1 (New England Biolabs). ..

    Purification:

    Article Title: ‘Shotgun DNA synthesis’ for the high-throughput construction of large DNA molecules
    Article Snippet: .. For each pool, restriction enzyme digestion was carried out by incubating 2 µl EarI (or EcoP15I), 5 µl NEB buffer 1, 0.5 µl 100× BSA and 10 µl water with 30 µl of purified (and pooled) shotgun synthesis fragments at 37°C for 3 h. For EcoP15I (NEB, Ipswich, MA, USA) restriction enzyme digestion, we used NEB buffer 3 and added 10× ATP. ..

    Incubation:

    Article Title: Dissecting super-enhancer hierarchy based on chromatin interactions
    Article Snippet: .. The annealed oligos were cloned into pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro vector (Addgene ID: 71236) using a Golden Gate Assembly strategy including: 100 ng of circular pLV plasmid, 0.2 μM annealed oligos, 2.1 buffer (1×) (New England Biolabs), 20 U of BsmBI restriction enzyme, 0.2 mM ATP, 0.1 mg/ml BSA, and 750 U of T4 DNA ligase (New England Biolabs) with the cycling parameters of 20 cycles of 37 °C for 5 min, 20 °C for 5 min; followed by 80 °C incubation for 20 min. Then K562 cells were transduced with lentivirus to stably express dCa s9-KRAB and sgRNA. .. Briefly, sequence-specific sgRNAs for site-specific interference of genomic targets were designed following described guidelines, and sequences were selected to minimize off-target effect based on publicly available filtering tools ( http://crispr.mit.edu/ ).

    Article Title: A Simple Procedure for Creating Scalable Phenotypic Screening Assays in Human Neurons
    Article Snippet: .. Individual plasmid DNA were incubated with appropriate restriction enzymes (New England Biolabs, Ipswich, MA) and NE Buffer (cat. no. B7201, NEB) for 1 h. Purple loading dye (cat. no. B7024S, NEB) was added to each sample which were loaded carefully on MiniReady Agarose Gel prepared in 1XTBE (cat. no. 101-3004, Biorad) alongside with Quick load purple 1 kb DNA ladder as a reference standard (cat. no. N0552G, NEB). .. The results demonstrated appropriate digest products for each plasmid as well as the maxi-prep RFLP and can be found in the supplemental data (Figure ).

    Article Title: The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components
    Article Snippet: .. Biochemical R.HpyAXII endonuclease activity assay One microliter of freshly prepared soluble fraction obtained from E. coli BL21(DE3) expressing R.HpyAXII was incubated for 3 h at 37°C with 500 ng E. coli ER2925 genomic DNA or pET15b plasmid DNA isolated from E. coli DH10B in presence of NEB buffer 1 (New England Biolabs). ..

    Activity Assay:

    Article Title: The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components
    Article Snippet: .. Biochemical R.HpyAXII endonuclease activity assay One microliter of freshly prepared soluble fraction obtained from E. coli BL21(DE3) expressing R.HpyAXII was incubated for 3 h at 37°C with 500 ng E. coli ER2925 genomic DNA or pET15b plasmid DNA isolated from E. coli DH10B in presence of NEB buffer 1 (New England Biolabs). ..

    Expressing:

    Article Title: The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components
    Article Snippet: .. Biochemical R.HpyAXII endonuclease activity assay One microliter of freshly prepared soluble fraction obtained from E. coli BL21(DE3) expressing R.HpyAXII was incubated for 3 h at 37°C with 500 ng E. coli ER2925 genomic DNA or pET15b plasmid DNA isolated from E. coli DH10B in presence of NEB buffer 1 (New England Biolabs). ..

    Plasmid Preparation:

    Article Title: Dissecting super-enhancer hierarchy based on chromatin interactions
    Article Snippet: .. The annealed oligos were cloned into pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro vector (Addgene ID: 71236) using a Golden Gate Assembly strategy including: 100 ng of circular pLV plasmid, 0.2 μM annealed oligos, 2.1 buffer (1×) (New England Biolabs), 20 U of BsmBI restriction enzyme, 0.2 mM ATP, 0.1 mg/ml BSA, and 750 U of T4 DNA ligase (New England Biolabs) with the cycling parameters of 20 cycles of 37 °C for 5 min, 20 °C for 5 min; followed by 80 °C incubation for 20 min. Then K562 cells were transduced with lentivirus to stably express dCa s9-KRAB and sgRNA. .. Briefly, sequence-specific sgRNAs for site-specific interference of genomic targets were designed following described guidelines, and sequences were selected to minimize off-target effect based on publicly available filtering tools ( http://crispr.mit.edu/ ).

    Article Title: A Simple Procedure for Creating Scalable Phenotypic Screening Assays in Human Neurons
    Article Snippet: .. Individual plasmid DNA were incubated with appropriate restriction enzymes (New England Biolabs, Ipswich, MA) and NE Buffer (cat. no. B7201, NEB) for 1 h. Purple loading dye (cat. no. B7024S, NEB) was added to each sample which were loaded carefully on MiniReady Agarose Gel prepared in 1XTBE (cat. no. 101-3004, Biorad) alongside with Quick load purple 1 kb DNA ladder as a reference standard (cat. no. N0552G, NEB). .. The results demonstrated appropriate digest products for each plasmid as well as the maxi-prep RFLP and can be found in the supplemental data (Figure ).

    Article Title: The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components
    Article Snippet: .. Biochemical R.HpyAXII endonuclease activity assay One microliter of freshly prepared soluble fraction obtained from E. coli BL21(DE3) expressing R.HpyAXII was incubated for 3 h at 37°C with 500 ng E. coli ER2925 genomic DNA or pET15b plasmid DNA isolated from E. coli DH10B in presence of NEB buffer 1 (New England Biolabs). ..

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    Monarch Plasmid Wash Buffer 1 54 ml
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    New England Biolabs methyltransferase buffer
    Single-Molecule Detection of CMG Pausing at a Lagging-Strand <t>Methyltransferase</t> Block (A) Schematic representation of experimental approach used in single-molecule DNA unwinding assays. (B) Images of a sample field of view showing accumulation of EGFP-RPA fluorescence signal at different time points from the addition of EGFP-RPA into the chamber. (C) Example unwinding traces of DNA substrates without a protein barrier. Traces exhibit a signal drop upon completion of unwinding due to dissociation of the leading-strand template (depicted in A). (D) Distribution of average fork rates measured in fully unwound substrates without MH (black) and after bypassing MH Lag (blue). Number of molecules are n(-MH) = 199, n(MH Lag after pause) = 20. (E) Sample unwinding traces of DNA substrates modified with MH Lag . Pausing observed at 800 bp is highlighted with gray rectangle. (F) Distribution of pause durations observed in molecules exhibiting a pausing event (n = 109). The solid line is a fit to a single exponential. See also Figure S6 .
    Methyltransferase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    New England Biolabs caspase buffer
    Relative activity of <t>caspase-8</t> with model peptides containing serine or phosphoserine at each position from P4 to P3′. Peptides modeled after the caspase-8 cleavage site of pro-caspase-3 were incubated with caspase-8 (100 n m ) for 5 min at 37 °C before the reaction was terminated by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. Means of the caspase activities toward the phosphorylated and the corresponding nonphosphorylated peptide were compared using analysis of variance and Tukey's test. All pairs had p values
    Caspase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    New England Biolabs neb bamhi buffer
    Variant M91V/E156K digestion of plasmid substrate pUC-GCT. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1 × <t>NEB</t> <t>BamHI</t> buffer. (−) indicates no enzyme addition.
    Neb Bamhi Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Single-Molecule Detection of CMG Pausing at a Lagging-Strand Methyltransferase Block (A) Schematic representation of experimental approach used in single-molecule DNA unwinding assays. (B) Images of a sample field of view showing accumulation of EGFP-RPA fluorescence signal at different time points from the addition of EGFP-RPA into the chamber. (C) Example unwinding traces of DNA substrates without a protein barrier. Traces exhibit a signal drop upon completion of unwinding due to dissociation of the leading-strand template (depicted in A). (D) Distribution of average fork rates measured in fully unwound substrates without MH (black) and after bypassing MH Lag (blue). Number of molecules are n(-MH) = 199, n(MH Lag after pause) = 20. (E) Sample unwinding traces of DNA substrates modified with MH Lag . Pausing observed at 800 bp is highlighted with gray rectangle. (F) Distribution of pause durations observed in molecules exhibiting a pausing event (n = 109). The solid line is a fit to a single exponential. See also Figure S6 .

    Journal: Cell Reports

    Article Title: Dynamics of the Eukaryotic Replicative Helicase at Lagging-Strand Protein Barriers Support the Steric Exclusion Model

    doi: 10.1016/j.celrep.2019.01.086

    Figure Lengend Snippet: Single-Molecule Detection of CMG Pausing at a Lagging-Strand Methyltransferase Block (A) Schematic representation of experimental approach used in single-molecule DNA unwinding assays. (B) Images of a sample field of view showing accumulation of EGFP-RPA fluorescence signal at different time points from the addition of EGFP-RPA into the chamber. (C) Example unwinding traces of DNA substrates without a protein barrier. Traces exhibit a signal drop upon completion of unwinding due to dissociation of the leading-strand template (depicted in A). (D) Distribution of average fork rates measured in fully unwound substrates without MH (black) and after bypassing MH Lag (blue). Number of molecules are n(-MH) = 199, n(MH Lag after pause) = 20. (E) Sample unwinding traces of DNA substrates modified with MH Lag . Pausing observed at 800 bp is highlighted with gray rectangle. (F) Distribution of pause durations observed in molecules exhibiting a pausing event (n = 109). The solid line is a fit to a single exponential. See also Figure S6 .

    Article Snippet: 5FdC-modified fork templates were mixed with HpaII methyltransferase (M.HpaII) in 1:4 DNA to protein molar ratio in methyltransferase buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM 2-mercaptoethanol (β-ME), 10 mM EDTA, NEB) supplemented with 100 μM S-adenosylmethionine (NEB), and incubated at 37°C for 3 hours.

    Techniques: Blocking Assay, Recombinase Polymerase Amplification, Fluorescence, Modification

    Relative activity of caspase-8 with model peptides containing serine or phosphoserine at each position from P4 to P3′. Peptides modeled after the caspase-8 cleavage site of pro-caspase-3 were incubated with caspase-8 (100 n m ) for 5 min at 37 °C before the reaction was terminated by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. Means of the caspase activities toward the phosphorylated and the corresponding nonphosphorylated peptide were compared using analysis of variance and Tukey's test. All pairs had p values

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation *

    doi: 10.1074/mcp.M113.037374

    Figure Lengend Snippet: Relative activity of caspase-8 with model peptides containing serine or phosphoserine at each position from P4 to P3′. Peptides modeled after the caspase-8 cleavage site of pro-caspase-3 were incubated with caspase-8 (100 n m ) for 5 min at 37 °C before the reaction was terminated by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. Means of the caspase activities toward the phosphorylated and the corresponding nonphosphorylated peptide were compared using analysis of variance and Tukey's test. All pairs had p values

    Article Snippet: After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Techniques: Activity Assay, Incubation, Standard Deviation

    Relative activities of caspase-3 and caspase-7 with model and phosphopeptides containing serine residues positioned within the P5–P4′ consensus motif for caspase cleavage. Caspase cleavage of peptide substrates was measured using fluorescence of an internally quenched tryptophan residue at 355 nm after excitation at 280 nm. Peptides or phosphopeptides modeled after Golgin-160 ( A ) and PARP1 ( B ) were assayed with caspase-3 or caspase-7 for 10 min at 37 °C and stopped by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. A , means of the caspase activities toward phosphorylated peptides and their nonphosphorylated counterparts were compared using analysis of variance and Tukey's test. For caspase-3, P4, P3, P1′, P2′, P3′, and P4′ phosphopeptide versus nonphosphorylated peptide pairs had p values

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation *

    doi: 10.1074/mcp.M113.037374

    Figure Lengend Snippet: Relative activities of caspase-3 and caspase-7 with model and phosphopeptides containing serine residues positioned within the P5–P4′ consensus motif for caspase cleavage. Caspase cleavage of peptide substrates was measured using fluorescence of an internally quenched tryptophan residue at 355 nm after excitation at 280 nm. Peptides or phosphopeptides modeled after Golgin-160 ( A ) and PARP1 ( B ) were assayed with caspase-3 or caspase-7 for 10 min at 37 °C and stopped by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. A , means of the caspase activities toward phosphorylated peptides and their nonphosphorylated counterparts were compared using analysis of variance and Tukey's test. For caspase-3, P4, P3, P1′, P2′, P3′, and P4′ phosphopeptide versus nonphosphorylated peptide pairs had p values

    Article Snippet: After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Techniques: Fluorescence, Standard Deviation

    Summary of the peptides identified via TAILS. A , a WebLogo analysis ( 18 ) of the 57 peptides with P1 aspartic acid that were identified via TAILS. B , a log 2 histogram of the quantified P1 Asp peptides was plotted to visualize the distribution of the effect of phosphorylation on caspase cleavage. The number of peptides in each group is indicated. C , a log 2 mountain plot of the quantified P1 Asp peptides. MST3, Yap1, and Golgin-160 are highlighted in red. The peptides and their relative abundance are also presented in supplemental Table S1 . D , E , Gene Ontology (GO) analysis was performed for all P1 Asp peptides ( D ) or those positively regulated by phosphorylation ( i.e. substrates with more pronounced regulation than MST3; see red bar with log 2

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation *

    doi: 10.1074/mcp.M113.037374

    Figure Lengend Snippet: Summary of the peptides identified via TAILS. A , a WebLogo analysis ( 18 ) of the 57 peptides with P1 aspartic acid that were identified via TAILS. B , a log 2 histogram of the quantified P1 Asp peptides was plotted to visualize the distribution of the effect of phosphorylation on caspase cleavage. The number of peptides in each group is indicated. C , a log 2 mountain plot of the quantified P1 Asp peptides. MST3, Yap1, and Golgin-160 are highlighted in red. The peptides and their relative abundance are also presented in supplemental Table S1 . D , E , Gene Ontology (GO) analysis was performed for all P1 Asp peptides ( D ) or those positively regulated by phosphorylation ( i.e. substrates with more pronounced regulation than MST3; see red bar with log 2

    Article Snippet: After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Techniques:

    Workflow for the global, unbiased analysis of the integration of phosphorylation and caspase-mediated degradation. A , illustration of the cleavage site nomenclature for proteases. Caspases cleave the scissile bond between a P1 aspartic acid and the P1′ residue. B , HeLa cell lysates were treated with or without λ phosphatase and subjected to caspase treatment followed by dephosphorylation of the sample previously left phosphorylated. Primary amines on protein N termini and lysine residues were dimethylated using heavy (+34, open circles) or light (+28, black circles) formaldehyde. Samples were pooled and trypsinized, which exposed an amine on the N terminus of the internal tryptic peptide. These peptides are captured through reaction with an ∼80-kDa aldehyde-substituted polymer. Importantly, native protein N termini and neo-N termini generated by caspase cleavage are resistant to reaction with the polymer because their reactive amines have been blocked by dimethylation. Enrichment of the N-terminome then occurs via negative selection when the reacted polymer is filtered away using a 10-kDa cut-off spin column. LC-MS/MS analysis of isotopically dimethylated peptides then allows comparative analysis between caspase degradomes of phosphorylated and dephosphorylated lysates. Caspase substrates will be inferred through identification of those peptides with a P1 aspartic acid. In the event that there is no difference in caspase substrate proteolysis between phosphorylated and dephosphorylated samples, a peptide ratio of ∼1:1 will be observed in MS1 [1]. Of interest are those peptide pairs that deviate from a 1:1 ratio [2].

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation *

    doi: 10.1074/mcp.M113.037374

    Figure Lengend Snippet: Workflow for the global, unbiased analysis of the integration of phosphorylation and caspase-mediated degradation. A , illustration of the cleavage site nomenclature for proteases. Caspases cleave the scissile bond between a P1 aspartic acid and the P1′ residue. B , HeLa cell lysates were treated with or without λ phosphatase and subjected to caspase treatment followed by dephosphorylation of the sample previously left phosphorylated. Primary amines on protein N termini and lysine residues were dimethylated using heavy (+34, open circles) or light (+28, black circles) formaldehyde. Samples were pooled and trypsinized, which exposed an amine on the N terminus of the internal tryptic peptide. These peptides are captured through reaction with an ∼80-kDa aldehyde-substituted polymer. Importantly, native protein N termini and neo-N termini generated by caspase cleavage are resistant to reaction with the polymer because their reactive amines have been blocked by dimethylation. Enrichment of the N-terminome then occurs via negative selection when the reacted polymer is filtered away using a 10-kDa cut-off spin column. LC-MS/MS analysis of isotopically dimethylated peptides then allows comparative analysis between caspase degradomes of phosphorylated and dephosphorylated lysates. Caspase substrates will be inferred through identification of those peptides with a P1 aspartic acid. In the event that there is no difference in caspase substrate proteolysis between phosphorylated and dephosphorylated samples, a peptide ratio of ∼1:1 will be observed in MS1 [1]. Of interest are those peptide pairs that deviate from a 1:1 ratio [2].

    Article Snippet: After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Techniques: De-Phosphorylation Assay, Generated, Selection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    TAILS analysis revealed MST3, Golgin-160, and Yap1 as validated candidates in which cleavage is regulated by phosphorylation. A , caspase recognition motif in MST3, Yap1, and Golgin-160. MS/MS analysis of MST3 ( B ), Yap1 ( C ), and Golgin-160 ( D ). E , HeLa lysates were treated identically to those samples prepared for N-terminomic analysis (as described in “Experimental Procedures”), except when they were utilized for Western blotting with antibodies directed against MST3, Yap1, or Golgin-160 as indicated. Caspase-3 and caspase-7 were used at concentrations of 0, 50, 500, and 5000 n m . The asterisk denotes a nonspecific band, and the arrow and arrowhead bands were differentially regulated by lysate dephosphorylation. F , HeLa cells were treated for 3 h with 1 μ m staurosporine and/or 500 n m okadaic acid prior to lysis. Lysates were analyzed using immunoblots with the indicated antibodies. PARP1 cleavage was used as a control for caspase activation. The asterisk denotes a nonspecific band.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation *

    doi: 10.1074/mcp.M113.037374

    Figure Lengend Snippet: TAILS analysis revealed MST3, Golgin-160, and Yap1 as validated candidates in which cleavage is regulated by phosphorylation. A , caspase recognition motif in MST3, Yap1, and Golgin-160. MS/MS analysis of MST3 ( B ), Yap1 ( C ), and Golgin-160 ( D ). E , HeLa lysates were treated identically to those samples prepared for N-terminomic analysis (as described in “Experimental Procedures”), except when they were utilized for Western blotting with antibodies directed against MST3, Yap1, or Golgin-160 as indicated. Caspase-3 and caspase-7 were used at concentrations of 0, 50, 500, and 5000 n m . The asterisk denotes a nonspecific band, and the arrow and arrowhead bands were differentially regulated by lysate dephosphorylation. F , HeLa cells were treated for 3 h with 1 μ m staurosporine and/or 500 n m okadaic acid prior to lysis. Lysates were analyzed using immunoblots with the indicated antibodies. PARP1 cleavage was used as a control for caspase activation. The asterisk denotes a nonspecific band.

    Article Snippet: After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Techniques: Mass Spectrometry, Western Blot, De-Phosphorylation Assay, Lysis, Activation Assay

    Caspase activities toward synthetic peptides modeled after substrates identified via TAILS. Synthetic peptides or phosphopeptides modeled after Yap1 ( A ), Golgin-160 ( B ), or MST3 ( C ) were treated with or without λ phosphatase as indicated prior to incubation with caspase-3 or caspase-7. Fluorescence of an internally quenched tryptophan residue at 355 nm after excitation at 280 nm was measured to assess caspase cleavage of peptide substrates. Error bars represent the standard deviation of four reactions. Means for the caspase activities with phosphorylated peptides and their unphosphorylated counterparts were compared using analysis of variance and Tukey's test. Control versus phosphatase-treated pairs for phosphopeptides all had p values

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation *

    doi: 10.1074/mcp.M113.037374

    Figure Lengend Snippet: Caspase activities toward synthetic peptides modeled after substrates identified via TAILS. Synthetic peptides or phosphopeptides modeled after Yap1 ( A ), Golgin-160 ( B ), or MST3 ( C ) were treated with or without λ phosphatase as indicated prior to incubation with caspase-3 or caspase-7. Fluorescence of an internally quenched tryptophan residue at 355 nm after excitation at 280 nm was measured to assess caspase cleavage of peptide substrates. Error bars represent the standard deviation of four reactions. Means for the caspase activities with phosphorylated peptides and their unphosphorylated counterparts were compared using analysis of variance and Tukey's test. Control versus phosphatase-treated pairs for phosphopeptides all had p values

    Article Snippet: After buffer exchange, the sample was split in two, diluted in 10× phosphatase buffer (NEB) and 5× caspase buffer (see above for the 1× recipe), and then treated with or without λ phosphatase (10 U NEB λ phosphatase per microgram of lysate) for 1 h at 37 °C.

    Techniques: Incubation, Fluorescence, Standard Deviation

    Variant M91V/E156K digestion of plasmid substrate pUC-GCT. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1 × NEB BamHI buffer. (−) indicates no enzyme addition.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Variant M91V/E156K digestion of plasmid substrate pUC-GCT. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1 × NEB BamHI buffer. (−) indicates no enzyme addition.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Plasmid Preparation, Incubation

    Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Plasmid Preparation, Derivative Assay, Incubation

    Digestion of T7 genomic DNA by variant M91V/E156K. ( A ) A virtual digest of T7 DNA using NEBcutter ( 29 ). Since site 5′-GCGTCCGC-3′ is nicked by this enzyme it was not included in the virtual digest. M is a size marker lane. ( B ) Actual results of digesting T7 DNA in 1× NEB BamHI buffer for various times at 37°C. A 30-fold molar excess of enzyme was used in each reaction. ( C ) A control showing no digestion of T7 DNA by 200 U wt NotI.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Digestion of T7 genomic DNA by variant M91V/E156K. ( A ) A virtual digest of T7 DNA using NEBcutter ( 29 ). Since site 5′-GCGTCCGC-3′ is nicked by this enzyme it was not included in the virtual digest. M is a size marker lane. ( B ) Actual results of digesting T7 DNA in 1× NEB BamHI buffer for various times at 37°C. A 30-fold molar excess of enzyme was used in each reaction. ( C ) A control showing no digestion of T7 DNA by 200 U wt NotI.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Marker

    Competitive cleavage of two linear substrates by variant E156K/M91V. The 2686 bp substrate was derived from pUC-GCT and the 1778 bp substrate was derived from pUC-NotI. Equimolar amounts of each substrate were mixed and incubated with increasing concentrations of enzyme. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer. (−) indicates no enzyme addition.

    Journal: Nucleic Acids Research

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    doi: 10.1093/nar/gkj483

    Figure Lengend Snippet: Competitive cleavage of two linear substrates by variant E156K/M91V. The 2686 bp substrate was derived from pUC-GCT and the 1778 bp substrate was derived from pUC-NotI. Equimolar amounts of each substrate were mixed and incubated with increasing concentrations of enzyme. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer. (−) indicates no enzyme addition.

    Article Snippet: This digestion and all subsequent reactions were carried out at 37°C in NEB BamHI buffer [150 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 1 mM DTT (pH 7.9 at 25°C)].

    Techniques: Variant Assay, Derivative Assay, Incubation