neb buffer 2 1  (New England Biolabs)


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    Structured Review

    New England Biolabs neb buffer 2 1
    TagI activity assays on (A) 5hm C and 5m C containing PCR products and (B,C) modified phage DNA in vitro and in vivo . ( A ) One μg of mixed PCR DNA (∼12 nM) made from modified dNTP mixtures was digested at 37°C for 1 h with 1 μg TagI (∼0.3 μM) in 2-fold serial dilutions in <t>NEB</t> buffer 2.1. The substrates for TagI digestion in 10 mM Mg 2+ contained C (3 kb) and 5hm C or 5m C (2.2 kb). The assay in 1 mM Mn 2+ was performed on C (3 kb), 5hm C (2.2 kb) and 5m C (1.2 kb) containing PCR DNA. The amount of TagI (μg) shown on top of each lane corresponds to 295, 147, 74, 37, and 18 nM of protein dimer, respectively. ( B ) Modified DNA from phage T4gt ( 5hm C, ∼0.2 nM), T4 ( g5hm C, ∼0.2 nM) or XP12 ( 5m C, 0.5 nM) was digested by TagI (∼0.3 μM) and control enzymes: tolerant to the presence of modified cytosines (MluCI (/AATT), 10 U), inhibited by cytosine modifications (HpaII (C/CGG), 10 U) and affected only by the presence of g5hm C (MspJI, 5U). ( C ) Late-log phase host cells were plated on soft agar to form a cell lawn, and diluted phages (Lambda, T4gt or T4) were spotted onto the cell lawns. Cell lysis and plaque formation indicated susceptibility to phage infection. No plaque formation indicated the restriction of T4gt phage by TagI expressing cells.
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    Images

    1) Product Images from "Crystal structure of the modification-dependent SRA-HNH endonuclease TagI"

    Article Title: Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky781

    TagI activity assays on (A) 5hm C and 5m C containing PCR products and (B,C) modified phage DNA in vitro and in vivo . ( A ) One μg of mixed PCR DNA (∼12 nM) made from modified dNTP mixtures was digested at 37°C for 1 h with 1 μg TagI (∼0.3 μM) in 2-fold serial dilutions in NEB buffer 2.1. The substrates for TagI digestion in 10 mM Mg 2+ contained C (3 kb) and 5hm C or 5m C (2.2 kb). The assay in 1 mM Mn 2+ was performed on C (3 kb), 5hm C (2.2 kb) and 5m C (1.2 kb) containing PCR DNA. The amount of TagI (μg) shown on top of each lane corresponds to 295, 147, 74, 37, and 18 nM of protein dimer, respectively. ( B ) Modified DNA from phage T4gt ( 5hm C, ∼0.2 nM), T4 ( g5hm C, ∼0.2 nM) or XP12 ( 5m C, 0.5 nM) was digested by TagI (∼0.3 μM) and control enzymes: tolerant to the presence of modified cytosines (MluCI (/AATT), 10 U), inhibited by cytosine modifications (HpaII (C/CGG), 10 U) and affected only by the presence of g5hm C (MspJI, 5U). ( C ) Late-log phase host cells were plated on soft agar to form a cell lawn, and diluted phages (Lambda, T4gt or T4) were spotted onto the cell lawns. Cell lysis and plaque formation indicated susceptibility to phage infection. No plaque formation indicated the restriction of T4gt phage by TagI expressing cells.
    Figure Legend Snippet: TagI activity assays on (A) 5hm C and 5m C containing PCR products and (B,C) modified phage DNA in vitro and in vivo . ( A ) One μg of mixed PCR DNA (∼12 nM) made from modified dNTP mixtures was digested at 37°C for 1 h with 1 μg TagI (∼0.3 μM) in 2-fold serial dilutions in NEB buffer 2.1. The substrates for TagI digestion in 10 mM Mg 2+ contained C (3 kb) and 5hm C or 5m C (2.2 kb). The assay in 1 mM Mn 2+ was performed on C (3 kb), 5hm C (2.2 kb) and 5m C (1.2 kb) containing PCR DNA. The amount of TagI (μg) shown on top of each lane corresponds to 295, 147, 74, 37, and 18 nM of protein dimer, respectively. ( B ) Modified DNA from phage T4gt ( 5hm C, ∼0.2 nM), T4 ( g5hm C, ∼0.2 nM) or XP12 ( 5m C, 0.5 nM) was digested by TagI (∼0.3 μM) and control enzymes: tolerant to the presence of modified cytosines (MluCI (/AATT), 10 U), inhibited by cytosine modifications (HpaII (C/CGG), 10 U) and affected only by the presence of g5hm C (MspJI, 5U). ( C ) Late-log phase host cells were plated on soft agar to form a cell lawn, and diluted phages (Lambda, T4gt or T4) were spotted onto the cell lawns. Cell lysis and plaque formation indicated susceptibility to phage infection. No plaque formation indicated the restriction of T4gt phage by TagI expressing cells.

    Techniques Used: Activity Assay, Polymerase Chain Reaction, Modification, In Vitro, In Vivo, Lysis, Infection, Expressing

    2) Product Images from "Crystal structure of the modification-dependent SRA-HNH endonuclease TagI"

    Article Title: Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky781

    TagI activity assays on (A) 5hm C and 5m C containing PCR products and (B,C) modified phage DNA in vitro and in vivo . ( A ) One μg of mixed PCR DNA (∼12 nM) made from modified dNTP mixtures was digested at 37°C for 1 h with 1 μg TagI (∼0.3 μM) in 2-fold serial dilutions in NEB buffer 2.1. The substrates for TagI digestion in 10 mM Mg 2+ contained C (3 kb) and 5hm C or 5m C (2.2 kb). The assay in 1 mM Mn 2+ was performed on C (3 kb), 5hm C (2.2 kb) and 5m C (1.2 kb) containing PCR DNA. The amount of TagI (μg) shown on top of each lane corresponds to 295, 147, 74, 37, and 18 nM of protein dimer, respectively. ( B ) Modified DNA from phage T4gt ( 5hm C, ∼0.2 nM), T4 ( g5hm C, ∼0.2 nM) or XP12 ( 5m C, 0.5 nM) was digested by TagI (∼0.3 μM) and control enzymes: tolerant to the presence of modified cytosines (MluCI (/AATT), 10 U), inhibited by cytosine modifications (HpaII (C/CGG), 10 U) and affected only by the presence of g5hm C (MspJI, 5U). ( C ) Late-log phase host cells were plated on soft agar to form a cell lawn, and diluted phages (Lambda, T4gt or T4) were spotted onto the cell lawns. Cell lysis and plaque formation indicated susceptibility to phage infection. No plaque formation indicated the restriction of T4gt phage by TagI expressing cells.
    Figure Legend Snippet: TagI activity assays on (A) 5hm C and 5m C containing PCR products and (B,C) modified phage DNA in vitro and in vivo . ( A ) One μg of mixed PCR DNA (∼12 nM) made from modified dNTP mixtures was digested at 37°C for 1 h with 1 μg TagI (∼0.3 μM) in 2-fold serial dilutions in NEB buffer 2.1. The substrates for TagI digestion in 10 mM Mg 2+ contained C (3 kb) and 5hm C or 5m C (2.2 kb). The assay in 1 mM Mn 2+ was performed on C (3 kb), 5hm C (2.2 kb) and 5m C (1.2 kb) containing PCR DNA. The amount of TagI (μg) shown on top of each lane corresponds to 295, 147, 74, 37, and 18 nM of protein dimer, respectively. ( B ) Modified DNA from phage T4gt ( 5hm C, ∼0.2 nM), T4 ( g5hm C, ∼0.2 nM) or XP12 ( 5m C, 0.5 nM) was digested by TagI (∼0.3 μM) and control enzymes: tolerant to the presence of modified cytosines (MluCI (/AATT), 10 U), inhibited by cytosine modifications (HpaII (C/CGG), 10 U) and affected only by the presence of g5hm C (MspJI, 5U). ( C ) Late-log phase host cells were plated on soft agar to form a cell lawn, and diluted phages (Lambda, T4gt or T4) were spotted onto the cell lawns. Cell lysis and plaque formation indicated susceptibility to phage infection. No plaque formation indicated the restriction of T4gt phage by TagI expressing cells.

    Techniques Used: Activity Assay, Polymerase Chain Reaction, Modification, In Vitro, In Vivo, Lysis, Infection, Expressing

    Efficacy of DNA TagI cleavage of substrates containing none to four 5m C bases. 20 ng (∼32 nM) of annealed 48-mer oligonucleotides were digested by TagI (0.125 μg, ∼92 nM of TagI dimer) in NEB buffer 2.1 for 30 min. Cleavage products were resolved in 15% urea-PAGE, stained by SYBR Gold and imaged on Typhoon imager. The predicted TagI cleavage site GCS/GC at the center of the duplexes conforms to the GC/NGC consensus for cleavage by Fnu4HI (20 U), which was used as a positive control. 5m Cs in oligoduplexes are represented by small black dots in the diagrams above each lane.
    Figure Legend Snippet: Efficacy of DNA TagI cleavage of substrates containing none to four 5m C bases. 20 ng (∼32 nM) of annealed 48-mer oligonucleotides were digested by TagI (0.125 μg, ∼92 nM of TagI dimer) in NEB buffer 2.1 for 30 min. Cleavage products were resolved in 15% urea-PAGE, stained by SYBR Gold and imaged on Typhoon imager. The predicted TagI cleavage site GCS/GC at the center of the duplexes conforms to the GC/NGC consensus for cleavage by Fnu4HI (20 U), which was used as a positive control. 5m Cs in oligoduplexes are represented by small black dots in the diagrams above each lane.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Positive Control

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    New England Biolabs nhoi
    <t>NhoI</t> endonuclease activity assays. ( A ) NhoI digestion of phage <t>XP12</t> (all m5 C), pBRFM (ScaI-linearized, three m5 C in G m5 CN G m5 CN G C), and T4gt DNA ( hm5 C). Enzyme dilution factors are indicated on the top of each lane. ( B ) A theoretical digest of pBRFM (NEBcutter) 25 by ScaI and another enzyme cleaving GCNGCNGC (expected sizes in bp: 1168, 966, 828, 515/504/484, 333/297, 153).
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    97
    New England Biolabs lambda phosphatase buffer
    Purification of a 245 kD Dam1p complex. (A) Purification of the Dam1p complex using a tagged Dad1p reveals 10 polypeptides. Purified Dam1p complex (as described in Materials and methods) and protein from an identical purification using an untagged control strain were separated on a 13.5% SDS-PAGE gel and silver stained. Polypeptides identified by mass spectrometric analysis of the complex are indicated. Those not yet identified are denoted by an asterisk. Bands labeled “background” are the highly homologous heat shock proteins Ssb1 and Ssb2 (66 kD) and Ssa1, Ssa2, Ssa3, and Ssa4 (70 kD). (B) Determination of S value and Stoke's radius of the Dam1p complex in yeast extracts. Sucrose gradient and Superose 6 gel filtration fractions (as described in Materials and methods) were probed with antibodies against Duo1p and Dam1p. The S value for Duo1p/Dam1p-containing complex was estimated as 6.5 from a linear fit of the S values versus peak fraction number of standards, and the Stoke's radius was estimated as 90.1 Å from a Porath correlation, relating to the elution volumes of the standard proteins to their known Stoke's radii ( Siegel and Monty, 1966 ). (C) Multiple subunits of the Dam1p complex are phosphorylated in vivo. Dam1p complex was purified in the presence of phosphatase inhibitors and then split into two aliquots, one of which was treated with <t>lambda</t> protein phosphatase. (D) Ask1p phosphorylation changes over the cell cycle. Ask1-GFP–tagged strains were grown to log phase and arrested in either α-factor (G 1 ), 0.2 M hydroxyurea (S phase), or by using temperature-sensitive cdc16–1 (metaphase) and cdc15–1 (telophase) mutants. Protein samples were run on an 8% SDS-PAGE gel and immunoblotted with antibodies against GFP.
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    90
    New England Biolabs proteinase k buffer
    Subcellular localization of Ynd1 proteins and E4orf4. A . ynd1Δ yeast cells expressing the indicated Ynd1 constructs were subjected to subcellular fractionation by differential centrifugation, and the presence of Ynd1 proteins in the various fractions was determined by a Western blot analysis. Rpb4 is a RNA polymerase II subunit which shuttles between the nucleus and the cytosol, and served here as a cytosolic fractionation marker. H: heavy membranes. L: light membranes. C: cytosol. B . ynd1Δ yeast cell extracts expressing vector control or WT Ynd1 together with E4orf4 were subjected to subcellular fractionation by differential centrifugation. Western blots were stained sequentially with antibodies recognizing tagged Ynd1, E4orf4, and the Tpd3 subunit of PP2A. C . Crude membranes were prepared from yeast cells expressing the indicated Ynd1 constructs or E4orf4, and were subjected to a <t>Proteinase</t> K protection assay in the presence or absence of Triton X-100. Proteins were analyzed by Western blots stained with the indicated antibodies. The Myc tag is fused to the amino terminus of Ynd1 constructs and the HA tag is fused to the cytosolic carboxyl tail. In this experiment, a longer exposure of the 518-Ynd1-containing blot allowed the presentation of a stronger signal in the blot to confirm that most of the protein was degraded by Proteinase K.
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    97
    New England Biolabs bst dna polymerase
    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the <t>Bst</t> <t>DNA</t> polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6
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    Image Search Results


    NhoI endonuclease activity assays. ( A ) NhoI digestion of phage XP12 (all m5 C), pBRFM (ScaI-linearized, three m5 C in G m5 CN G m5 CN G C), and T4gt DNA ( hm5 C). Enzyme dilution factors are indicated on the top of each lane. ( B ) A theoretical digest of pBRFM (NEBcutter) 25 by ScaI and another enzyme cleaving GCNGCNGC (expected sizes in bp: 1168, 966, 828, 515/504/484, 333/297, 153).

    Journal: Scientific Reports

    Article Title: Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes

    doi: 10.1038/srep28579

    Figure Lengend Snippet: NhoI endonuclease activity assays. ( A ) NhoI digestion of phage XP12 (all m5 C), pBRFM (ScaI-linearized, three m5 C in G m5 CN G m5 CN G C), and T4gt DNA ( hm5 C). Enzyme dilution factors are indicated on the top of each lane. ( B ) A theoretical digest of pBRFM (NEBcutter) 25 by ScaI and another enzyme cleaving GCNGCNGC (expected sizes in bp: 1168, 966, 828, 515/504/484, 333/297, 153).

    Article Snippet: Direct sequencing of XP12 DNA digested by NhoI (NEB buffer 2.1 and high enzyme concentration) also identified a star site Am5 CG G m5 C , consistent with the small digested fragments around 100 bp that constitute the final products.

    Techniques: Activity Assay

    Digestion of m5 C-modified duplex oligos (GCWGC site) by BisI or NhoI. The 5′-FAM-labeled top strand contains two m5 C bases (G m5 CAG m5 C) and the bottom strands contain either two (G m5 CTG m5 C), one (G m5 CTGC, internal C methylated), or no m5 C bases (GCTGC), respectively. Thus, the annealed oligos contain a total of four, three, or two m5 C. P1 (20 bp) and P2 (14 bp) are the cleavage products. P3 is a possible top-strand nicked intermediate (NI) due to asymmetric nicking of the top strand. The substrate (sub, 34 bp), P1, and P3 were detected by FAM fluorescence imaging. ( A , B ). BisI- and NhoI-digested duplex oligos (four, three, or two m5 C) analyzed on a 15% PAG-urea denaturing gel. C . Partial digestion of the duplex oligos by NhoI and the DNA products were analyzed on a 20% TBE (non-denaturing) gel, stained by SYBR Gold and imaged by fluorescence imaging. The 5-bp dsDNA size marker (Fermentas) and the single-stranded oligos (IDT, 20–100 nt) were used to estimate the size of the cleavage products.

    Journal: Scientific Reports

    Article Title: Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes

    doi: 10.1038/srep28579

    Figure Lengend Snippet: Digestion of m5 C-modified duplex oligos (GCWGC site) by BisI or NhoI. The 5′-FAM-labeled top strand contains two m5 C bases (G m5 CAG m5 C) and the bottom strands contain either two (G m5 CTG m5 C), one (G m5 CTGC, internal C methylated), or no m5 C bases (GCTGC), respectively. Thus, the annealed oligos contain a total of four, three, or two m5 C. P1 (20 bp) and P2 (14 bp) are the cleavage products. P3 is a possible top-strand nicked intermediate (NI) due to asymmetric nicking of the top strand. The substrate (sub, 34 bp), P1, and P3 were detected by FAM fluorescence imaging. ( A , B ). BisI- and NhoI-digested duplex oligos (four, three, or two m5 C) analyzed on a 15% PAG-urea denaturing gel. C . Partial digestion of the duplex oligos by NhoI and the DNA products were analyzed on a 20% TBE (non-denaturing) gel, stained by SYBR Gold and imaged by fluorescence imaging. The 5-bp dsDNA size marker (Fermentas) and the single-stranded oligos (IDT, 20–100 nt) were used to estimate the size of the cleavage products.

    Article Snippet: Direct sequencing of XP12 DNA digested by NhoI (NEB buffer 2.1 and high enzyme concentration) also identified a star site Am5 CG G m5 C , consistent with the small digested fragments around 100 bp that constitute the final products.

    Techniques: Modification, Labeling, CTG Assay, Methylation, Fluorescence, Imaging, Staining, Marker

    Run-off sequencing to determine NhoI cut sites. DNA run-off sequencing at G m5 CT G C (incubated with NhoI endonuclease, but this site not digested) and G m5 CA G m5 CA G C sites of NhoI-digested plasmid pBRFM (M.Fnu4HI). The G m5 CT G C site indicated by the black bar contains two m5 C. The G m5 CA G m5 CA G C site indicated by the blue bar contains three m5 C.

    Journal: Scientific Reports

    Article Title: Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes

    doi: 10.1038/srep28579

    Figure Lengend Snippet: Run-off sequencing to determine NhoI cut sites. DNA run-off sequencing at G m5 CT G C (incubated with NhoI endonuclease, but this site not digested) and G m5 CA G m5 CA G C sites of NhoI-digested plasmid pBRFM (M.Fnu4HI). The G m5 CT G C site indicated by the black bar contains two m5 C. The G m5 CA G m5 CA G C site indicated by the blue bar contains three m5 C.

    Article Snippet: Direct sequencing of XP12 DNA digested by NhoI (NEB buffer 2.1 and high enzyme concentration) also identified a star site Am5 CG G m5 C , consistent with the small digested fragments around 100 bp that constitute the final products.

    Techniques: Sequencing, Incubation, Plasmid Preparation

    Purification of a 245 kD Dam1p complex. (A) Purification of the Dam1p complex using a tagged Dad1p reveals 10 polypeptides. Purified Dam1p complex (as described in Materials and methods) and protein from an identical purification using an untagged control strain were separated on a 13.5% SDS-PAGE gel and silver stained. Polypeptides identified by mass spectrometric analysis of the complex are indicated. Those not yet identified are denoted by an asterisk. Bands labeled “background” are the highly homologous heat shock proteins Ssb1 and Ssb2 (66 kD) and Ssa1, Ssa2, Ssa3, and Ssa4 (70 kD). (B) Determination of S value and Stoke's radius of the Dam1p complex in yeast extracts. Sucrose gradient and Superose 6 gel filtration fractions (as described in Materials and methods) were probed with antibodies against Duo1p and Dam1p. The S value for Duo1p/Dam1p-containing complex was estimated as 6.5 from a linear fit of the S values versus peak fraction number of standards, and the Stoke's radius was estimated as 90.1 Å from a Porath correlation, relating to the elution volumes of the standard proteins to their known Stoke's radii ( Siegel and Monty, 1966 ). (C) Multiple subunits of the Dam1p complex are phosphorylated in vivo. Dam1p complex was purified in the presence of phosphatase inhibitors and then split into two aliquots, one of which was treated with lambda protein phosphatase. (D) Ask1p phosphorylation changes over the cell cycle. Ask1-GFP–tagged strains were grown to log phase and arrested in either α-factor (G 1 ), 0.2 M hydroxyurea (S phase), or by using temperature-sensitive cdc16–1 (metaphase) and cdc15–1 (telophase) mutants. Protein samples were run on an 8% SDS-PAGE gel and immunoblotted with antibodies against GFP.

    Journal: The Journal of Cell Biology

    Article Title: Implication of a novel multiprotein Dam1p complex in outer kinetochore function

    doi: 10.1083/jcb.200109063

    Figure Lengend Snippet: Purification of a 245 kD Dam1p complex. (A) Purification of the Dam1p complex using a tagged Dad1p reveals 10 polypeptides. Purified Dam1p complex (as described in Materials and methods) and protein from an identical purification using an untagged control strain were separated on a 13.5% SDS-PAGE gel and silver stained. Polypeptides identified by mass spectrometric analysis of the complex are indicated. Those not yet identified are denoted by an asterisk. Bands labeled “background” are the highly homologous heat shock proteins Ssb1 and Ssb2 (66 kD) and Ssa1, Ssa2, Ssa3, and Ssa4 (70 kD). (B) Determination of S value and Stoke's radius of the Dam1p complex in yeast extracts. Sucrose gradient and Superose 6 gel filtration fractions (as described in Materials and methods) were probed with antibodies against Duo1p and Dam1p. The S value for Duo1p/Dam1p-containing complex was estimated as 6.5 from a linear fit of the S values versus peak fraction number of standards, and the Stoke's radius was estimated as 90.1 Å from a Porath correlation, relating to the elution volumes of the standard proteins to their known Stoke's radii ( Siegel and Monty, 1966 ). (C) Multiple subunits of the Dam1p complex are phosphorylated in vivo. Dam1p complex was purified in the presence of phosphatase inhibitors and then split into two aliquots, one of which was treated with lambda protein phosphatase. (D) Ask1p phosphorylation changes over the cell cycle. Ask1-GFP–tagged strains were grown to log phase and arrested in either α-factor (G 1 ), 0.2 M hydroxyurea (S phase), or by using temperature-sensitive cdc16–1 (metaphase) and cdc15–1 (telophase) mutants. Protein samples were run on an 8% SDS-PAGE gel and immunoblotted with antibodies against GFP.

    Article Snippet: For studies on the dephosphorylated complex, protein bound to either the IgG sepharose or S protein agarose was washed into lambda phosphatase buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5 mM DTT, 0.01% Brij 35, 2 mM MnCl2 ) and incubated with 4 μl of lambda protein phosphatase (New England Biolabs, Inc.) for 1 h at 30°C.

    Techniques: Purification, SDS Page, Staining, Labeling, Filtration, In Vivo

    Subcellular localization of Ynd1 proteins and E4orf4. A . ynd1Δ yeast cells expressing the indicated Ynd1 constructs were subjected to subcellular fractionation by differential centrifugation, and the presence of Ynd1 proteins in the various fractions was determined by a Western blot analysis. Rpb4 is a RNA polymerase II subunit which shuttles between the nucleus and the cytosol, and served here as a cytosolic fractionation marker. H: heavy membranes. L: light membranes. C: cytosol. B . ynd1Δ yeast cell extracts expressing vector control or WT Ynd1 together with E4orf4 were subjected to subcellular fractionation by differential centrifugation. Western blots were stained sequentially with antibodies recognizing tagged Ynd1, E4orf4, and the Tpd3 subunit of PP2A. C . Crude membranes were prepared from yeast cells expressing the indicated Ynd1 constructs or E4orf4, and were subjected to a Proteinase K protection assay in the presence or absence of Triton X-100. Proteins were analyzed by Western blots stained with the indicated antibodies. The Myc tag is fused to the amino terminus of Ynd1 constructs and the HA tag is fused to the cytosolic carboxyl tail. In this experiment, a longer exposure of the 518-Ynd1-containing blot allowed the presentation of a stronger signal in the blot to confirm that most of the protein was degraded by Proteinase K.

    Journal: PLoS ONE

    Article Title: The Cytosolic Tail of the Golgi Apyrase Ynd1 Mediates E4orf4-Induced Toxicity in Saccharomyces cerevisiae

    doi: 10.1371/journal.pone.0015539

    Figure Lengend Snippet: Subcellular localization of Ynd1 proteins and E4orf4. A . ynd1Δ yeast cells expressing the indicated Ynd1 constructs were subjected to subcellular fractionation by differential centrifugation, and the presence of Ynd1 proteins in the various fractions was determined by a Western blot analysis. Rpb4 is a RNA polymerase II subunit which shuttles between the nucleus and the cytosol, and served here as a cytosolic fractionation marker. H: heavy membranes. L: light membranes. C: cytosol. B . ynd1Δ yeast cell extracts expressing vector control or WT Ynd1 together with E4orf4 were subjected to subcellular fractionation by differential centrifugation. Western blots were stained sequentially with antibodies recognizing tagged Ynd1, E4orf4, and the Tpd3 subunit of PP2A. C . Crude membranes were prepared from yeast cells expressing the indicated Ynd1 constructs or E4orf4, and were subjected to a Proteinase K protection assay in the presence or absence of Triton X-100. Proteins were analyzed by Western blots stained with the indicated antibodies. The Myc tag is fused to the amino terminus of Ynd1 constructs and the HA tag is fused to the cytosolic carboxyl tail. In this experiment, a longer exposure of the 518-Ynd1-containing blot allowed the presentation of a stronger signal in the blot to confirm that most of the protein was degraded by Proteinase K.

    Article Snippet: Proteinase K protection assay Crude membrane extracts were prepared as above, except for omitting the centrifugation step at 10,000 g. The pellet containing total cellular membranes was suspended in Proteinase K buffer (10 mM Triethanolamine pH 7.2, 0.8 M Sorbitol, 1 mM EDTA) and subsequently treated with one of the following: A) Proteinase K buffer; B) Proteinase K buffer +1% Triton X-100; C) Proteinase K buffer +200 µg/ml Proteinase K (New England BioLabs, Ipswitch, MA); D) Proteinase K buffer +1% Triton X-100+200 µg/ml Proteinase K. All reactions were incubated at 37°C for 45 minutes, and were stopped by addition of 100% TCA to the sample.

    Techniques: Expressing, Construct, Fractionation, Centrifugation, Western Blot, Marker, Plasmid Preparation, Staining

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Journal: Scientific Reports

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    doi: 10.1038/s41598-017-13881-4

    Figure Lengend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Article Snippet: This optimized reaction mixture (total, 25 μl) contained 12.5 μl 2 × Bst DNA polymerase (NEB, Ipswich, MA, USA) reaction buffer [1 × containing 1.6 mM dNTPs, 1 M betaine, 4 mM MgSO4 , 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2 SO4 , and 0.1% Triton X-20 (Sigma-Aldrich Inc., Saint Louis, USA), Double Helix Tech.

    Techniques: Amplification