neb buffer 2  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92

    Structured Review

    New England Biolabs neb buffer 2
    Purification of Bam HI catalytic mutant ( Bam HI*)-gHNH fusion enzyme and nicking activity assay in the presence of divalent cations. (A) Schematic diagram of Bam HI catalytic mutant ( Bam HI*) in fusion with phage gHNH nicking domain (the size of DNA and protein domains are not shown in scale). The diagram is simplified to show the monomeric fusion only. (B) SDS-PAGE analysis of partially purified Bam HI*-gHNH fusion protein. Lane 3, chitin column purified fusion protein (predicted molecular mass in ∼33.53 kDa). Following DTT cleavage overnight, eluants 1 and 2 from the chitin column were concentrated by low-speed centrifugation in centrifugal filter unit (Millipore, 10 kDa cut-off) and resuspended in an enzyme storage buffer. Approximately 20 and 5 μg of the Bam HI mutant and fusion proteins were loaded in the gel. MMS, protein molecular mass standard in Daltons (Da) <t>(NEB).</t> (C) A time course (5–60 min) of pUC19 nicking digestion (partial) by the fusion enzyme in NEB buffer 2 supplemented with MnCl 2 . Approximately 80% of supercoiled DNA was converted to nicked circular form after 1 h nicking reaction. A weak linear band also appeared. SC and NC, supercoiled and nicked circular DNA, respectively. 2-log, DNA size ladder (100 bp to 10 kb, NEB).
    Neb Buffer 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/neb buffer 2/product/New England Biolabs
    Average 92 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    neb buffer 2 - by Bioz Stars, 2022-09
    92/100 stars

    Images

    1) Product Images from "Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain"

    Article Title: Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.787073

    Purification of Bam HI catalytic mutant ( Bam HI*)-gHNH fusion enzyme and nicking activity assay in the presence of divalent cations. (A) Schematic diagram of Bam HI catalytic mutant ( Bam HI*) in fusion with phage gHNH nicking domain (the size of DNA and protein domains are not shown in scale). The diagram is simplified to show the monomeric fusion only. (B) SDS-PAGE analysis of partially purified Bam HI*-gHNH fusion protein. Lane 3, chitin column purified fusion protein (predicted molecular mass in ∼33.53 kDa). Following DTT cleavage overnight, eluants 1 and 2 from the chitin column were concentrated by low-speed centrifugation in centrifugal filter unit (Millipore, 10 kDa cut-off) and resuspended in an enzyme storage buffer. Approximately 20 and 5 μg of the Bam HI mutant and fusion proteins were loaded in the gel. MMS, protein molecular mass standard in Daltons (Da) (NEB). (C) A time course (5–60 min) of pUC19 nicking digestion (partial) by the fusion enzyme in NEB buffer 2 supplemented with MnCl 2 . Approximately 80% of supercoiled DNA was converted to nicked circular form after 1 h nicking reaction. A weak linear band also appeared. SC and NC, supercoiled and nicked circular DNA, respectively. 2-log, DNA size ladder (100 bp to 10 kb, NEB).
    Figure Legend Snippet: Purification of Bam HI catalytic mutant ( Bam HI*)-gHNH fusion enzyme and nicking activity assay in the presence of divalent cations. (A) Schematic diagram of Bam HI catalytic mutant ( Bam HI*) in fusion with phage gHNH nicking domain (the size of DNA and protein domains are not shown in scale). The diagram is simplified to show the monomeric fusion only. (B) SDS-PAGE analysis of partially purified Bam HI*-gHNH fusion protein. Lane 3, chitin column purified fusion protein (predicted molecular mass in ∼33.53 kDa). Following DTT cleavage overnight, eluants 1 and 2 from the chitin column were concentrated by low-speed centrifugation in centrifugal filter unit (Millipore, 10 kDa cut-off) and resuspended in an enzyme storage buffer. Approximately 20 and 5 μg of the Bam HI mutant and fusion proteins were loaded in the gel. MMS, protein molecular mass standard in Daltons (Da) (NEB). (C) A time course (5–60 min) of pUC19 nicking digestion (partial) by the fusion enzyme in NEB buffer 2 supplemented with MnCl 2 . Approximately 80% of supercoiled DNA was converted to nicked circular form after 1 h nicking reaction. A weak linear band also appeared. SC and NC, supercoiled and nicked circular DNA, respectively. 2-log, DNA size ladder (100 bp to 10 kb, NEB).

    Techniques Used: Purification, Mutagenesis, Activity Assay, SDS Page, Centrifugation

    DNA run-off sequencing to map the nicking site(s) and nicking distance from a Bam HI site in pUC19. The plasmid substrate was digested by the fusion nickase at 37°C for 30 min in NEB buffer 2 supplemented with 1 mM MnCl 2 . (A) The composite nicking site GGATCC-N6-AC↑CGA is shown in a schematic diagram. (B) Top-strand sequencing read from the nicked bottom strand. The up arrow indicates the bottom strand of AC↑CGA was nicked and the appearance of an extra adenine (A) peak in the doublet as “A/C.” The addition of an extra peak “A” was catalyzed by the Taq DNA polymerase (template-independent terminal nucleotide transferase) when the template DNA is broken (nicked). Del1 and Del2 indicate some intervening sequences were manually deleted to show the downstream sites (ACCGA and ACCGC). (C) Bottom-strand sequencing read form nicked top-strand DNA. A star nicking site 5′ GGATCC N5 AGTCG 3′ was detected by the presence of an “A/T” doublet. The same sequence was shown as a reverse-complement in (D) . The down arrow indicates the nicked position.
    Figure Legend Snippet: DNA run-off sequencing to map the nicking site(s) and nicking distance from a Bam HI site in pUC19. The plasmid substrate was digested by the fusion nickase at 37°C for 30 min in NEB buffer 2 supplemented with 1 mM MnCl 2 . (A) The composite nicking site GGATCC-N6-AC↑CGA is shown in a schematic diagram. (B) Top-strand sequencing read from the nicked bottom strand. The up arrow indicates the bottom strand of AC↑CGA was nicked and the appearance of an extra adenine (A) peak in the doublet as “A/C.” The addition of an extra peak “A” was catalyzed by the Taq DNA polymerase (template-independent terminal nucleotide transferase) when the template DNA is broken (nicked). Del1 and Del2 indicate some intervening sequences were manually deleted to show the downstream sites (ACCGA and ACCGC). (C) Bottom-strand sequencing read form nicked top-strand DNA. A star nicking site 5′ GGATCC N5 AGTCG 3′ was detected by the presence of an “A/T” doublet. The same sequence was shown as a reverse-complement in (D) . The down arrow indicates the nicked position.

    Techniques Used: Sequencing, Plasmid Preparation

    DNA run-off sequencing to map the nicking site and nicking distance from Bam HI in the engineered composite site GGATCC-N(4-5)-AC↑CGG in pUC19 (1xACCGG). Top-strand sequencing read using nicked bottom-strand template. DNA nicking reactions were carried out in NEB buffer 2 supplemented with Mn 2+ (1 mM) at 37°C for 30 min. The up arrows indicate the nicked position where a strong A/C doublet was present. Del1 indicates the intervening sequence had been manually deleted to show the downstream site ACCGA (where no nicking had occurred). The composite sites GGATCC N4 ACCGG and GGATCC N5 ACCGG were inserted into pUC19 multiple cloning sites and flanked by Hin dIII and Eco RI sites. In addition, a star site (AGTCG) upstream of the Bam HI site was changed to the sequence TATAG to eliminate the star nicking activity.
    Figure Legend Snippet: DNA run-off sequencing to map the nicking site and nicking distance from Bam HI in the engineered composite site GGATCC-N(4-5)-AC↑CGG in pUC19 (1xACCGG). Top-strand sequencing read using nicked bottom-strand template. DNA nicking reactions were carried out in NEB buffer 2 supplemented with Mn 2+ (1 mM) at 37°C for 30 min. The up arrows indicate the nicked position where a strong A/C doublet was present. Del1 indicates the intervening sequence had been manually deleted to show the downstream site ACCGA (where no nicking had occurred). The composite sites GGATCC N4 ACCGG and GGATCC N5 ACCGG were inserted into pUC19 multiple cloning sites and flanked by Hin dIII and Eco RI sites. In addition, a star site (AGTCG) upstream of the Bam HI site was changed to the sequence TATAG to eliminate the star nicking activity.

    Techniques Used: Sequencing, Clone Assay, Activity Assay

    DNA run-off sequencing of the nicking sites and nicking distance from the Bam HI site. (A) Top-strand sequencing read from nicked bottom strand. Nicking reactions were carried out in NEB buffer 2 supplemented with MnCl 2 (1 mM) or NiCl 2 (1 mM) using pUC19 (2xACCGG) containing the composite site 5′ CCGGT-N5-GGATCC-N5 ACCGG 3′. The duplex oligos containing the composite site was inserted into pUC19 and flanked by Hin dIII (AAGCTT) and Eco RI (GAATTC) sites. (B) Bottom-strand sequencing read (nicked top-strand template) near the Bam HI site. The composite site contains a nick as indicated by a strong A/C doublet. If the bottom strand is shown as a reverse-complement, a T/G doublet would be present (C) in the nicking site.
    Figure Legend Snippet: DNA run-off sequencing of the nicking sites and nicking distance from the Bam HI site. (A) Top-strand sequencing read from nicked bottom strand. Nicking reactions were carried out in NEB buffer 2 supplemented with MnCl 2 (1 mM) or NiCl 2 (1 mM) using pUC19 (2xACCGG) containing the composite site 5′ CCGGT-N5-GGATCC-N5 ACCGG 3′. The duplex oligos containing the composite site was inserted into pUC19 and flanked by Hin dIII (AAGCTT) and Eco RI (GAATTC) sites. (B) Bottom-strand sequencing read (nicked top-strand template) near the Bam HI site. The composite site contains a nick as indicated by a strong A/C doublet. If the bottom strand is shown as a reverse-complement, a T/G doublet would be present (C) in the nicking site.

    Techniques Used: Sequencing

    2) Product Images from "Expression and purification of a single-chain Type IV restriction enzyme Eco94GmrSD and determination of its substrate preference"

    Article Title: Expression and purification of a single-chain Type IV restriction enzyme Eco94GmrSD and determination of its substrate preference

    Journal: Scientific Reports

    doi: 10.1038/srep09747

    Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in NEB buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).
    Figure Legend Snippet: Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in NEB buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).

    Techniques Used: Polymerase Chain Reaction, Generated, Modification

    3) Product Images from "Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain"

    Article Title: Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.787073

    Purification of Bam HI catalytic mutant ( Bam HI*)-gHNH fusion enzyme and nicking activity assay in the presence of divalent cations. (A) Schematic diagram of Bam HI catalytic mutant ( Bam HI*) in fusion with phage gHNH nicking domain (the size of DNA and protein domains are not shown in scale). The diagram is simplified to show the monomeric fusion only. (B) SDS-PAGE analysis of partially purified Bam HI*-gHNH fusion protein. Lane 3, chitin column purified fusion protein (predicted molecular mass in ∼33.53 kDa). Following DTT cleavage overnight, eluants 1 and 2 from the chitin column were concentrated by low-speed centrifugation in centrifugal filter unit (Millipore, 10 kDa cut-off) and resuspended in an enzyme storage buffer. Approximately 20 and 5 μg of the Bam HI mutant and fusion proteins were loaded in the gel. MMS, protein molecular mass standard in Daltons (Da) (NEB). (C) A time course (5–60 min) of pUC19 nicking digestion (partial) by the fusion enzyme in NEB buffer 2 supplemented with MnCl 2 . Approximately 80% of supercoiled DNA was converted to nicked circular form after 1 h nicking reaction. A weak linear band also appeared. SC and NC, supercoiled and nicked circular DNA, respectively. 2-log, DNA size ladder (100 bp to 10 kb, NEB).
    Figure Legend Snippet: Purification of Bam HI catalytic mutant ( Bam HI*)-gHNH fusion enzyme and nicking activity assay in the presence of divalent cations. (A) Schematic diagram of Bam HI catalytic mutant ( Bam HI*) in fusion with phage gHNH nicking domain (the size of DNA and protein domains are not shown in scale). The diagram is simplified to show the monomeric fusion only. (B) SDS-PAGE analysis of partially purified Bam HI*-gHNH fusion protein. Lane 3, chitin column purified fusion protein (predicted molecular mass in ∼33.53 kDa). Following DTT cleavage overnight, eluants 1 and 2 from the chitin column were concentrated by low-speed centrifugation in centrifugal filter unit (Millipore, 10 kDa cut-off) and resuspended in an enzyme storage buffer. Approximately 20 and 5 μg of the Bam HI mutant and fusion proteins were loaded in the gel. MMS, protein molecular mass standard in Daltons (Da) (NEB). (C) A time course (5–60 min) of pUC19 nicking digestion (partial) by the fusion enzyme in NEB buffer 2 supplemented with MnCl 2 . Approximately 80% of supercoiled DNA was converted to nicked circular form after 1 h nicking reaction. A weak linear band also appeared. SC and NC, supercoiled and nicked circular DNA, respectively. 2-log, DNA size ladder (100 bp to 10 kb, NEB).

    Techniques Used: Purification, Mutagenesis, Activity Assay, SDS Page, Centrifugation

    DNA run-off sequencing to map the nicking site(s) and nicking distance from a Bam HI site in pUC19. The plasmid substrate was digested by the fusion nickase at 37°C for 30 min in NEB buffer 2 supplemented with 1 mM MnCl 2 . (A) The composite nicking site GGATCC-N6-AC↑CGA is shown in a schematic diagram. (B) Top-strand sequencing read from the nicked bottom strand. The up arrow indicates the bottom strand of AC↑CGA was nicked and the appearance of an extra adenine (A) peak in the doublet as “A/C.” The addition of an extra peak “A” was catalyzed by the Taq DNA polymerase (template-independent terminal nucleotide transferase) when the template DNA is broken (nicked). Del1 and Del2 indicate some intervening sequences were manually deleted to show the downstream sites (ACCGA and ACCGC). (C) Bottom-strand sequencing read form nicked top-strand DNA. A star nicking site 5′ GGATCC N5 AGTCG 3′ was detected by the presence of an “A/T” doublet. The same sequence was shown as a reverse-complement in (D) . The down arrow indicates the nicked position.
    Figure Legend Snippet: DNA run-off sequencing to map the nicking site(s) and nicking distance from a Bam HI site in pUC19. The plasmid substrate was digested by the fusion nickase at 37°C for 30 min in NEB buffer 2 supplemented with 1 mM MnCl 2 . (A) The composite nicking site GGATCC-N6-AC↑CGA is shown in a schematic diagram. (B) Top-strand sequencing read from the nicked bottom strand. The up arrow indicates the bottom strand of AC↑CGA was nicked and the appearance of an extra adenine (A) peak in the doublet as “A/C.” The addition of an extra peak “A” was catalyzed by the Taq DNA polymerase (template-independent terminal nucleotide transferase) when the template DNA is broken (nicked). Del1 and Del2 indicate some intervening sequences were manually deleted to show the downstream sites (ACCGA and ACCGC). (C) Bottom-strand sequencing read form nicked top-strand DNA. A star nicking site 5′ GGATCC N5 AGTCG 3′ was detected by the presence of an “A/T” doublet. The same sequence was shown as a reverse-complement in (D) . The down arrow indicates the nicked position.

    Techniques Used: Sequencing, Plasmid Preparation

    DNA run-off sequencing to map the nicking site and nicking distance from Bam HI in the engineered composite site GGATCC-N(4-5)-AC↑CGG in pUC19 (1xACCGG). Top-strand sequencing read using nicked bottom-strand template. DNA nicking reactions were carried out in NEB buffer 2 supplemented with Mn 2+ (1 mM) at 37°C for 30 min. The up arrows indicate the nicked position where a strong A/C doublet was present. Del1 indicates the intervening sequence had been manually deleted to show the downstream site ACCGA (where no nicking had occurred). The composite sites GGATCC N4 ACCGG and GGATCC N5 ACCGG were inserted into pUC19 multiple cloning sites and flanked by Hin dIII and Eco RI sites. In addition, a star site (AGTCG) upstream of the Bam HI site was changed to the sequence TATAG to eliminate the star nicking activity.
    Figure Legend Snippet: DNA run-off sequencing to map the nicking site and nicking distance from Bam HI in the engineered composite site GGATCC-N(4-5)-AC↑CGG in pUC19 (1xACCGG). Top-strand sequencing read using nicked bottom-strand template. DNA nicking reactions were carried out in NEB buffer 2 supplemented with Mn 2+ (1 mM) at 37°C for 30 min. The up arrows indicate the nicked position where a strong A/C doublet was present. Del1 indicates the intervening sequence had been manually deleted to show the downstream site ACCGA (where no nicking had occurred). The composite sites GGATCC N4 ACCGG and GGATCC N5 ACCGG were inserted into pUC19 multiple cloning sites and flanked by Hin dIII and Eco RI sites. In addition, a star site (AGTCG) upstream of the Bam HI site was changed to the sequence TATAG to eliminate the star nicking activity.

    Techniques Used: Sequencing, Clone Assay, Activity Assay

    DNA run-off sequencing of the nicking sites and nicking distance from the Bam HI site. (A) Top-strand sequencing read from nicked bottom strand. Nicking reactions were carried out in NEB buffer 2 supplemented with MnCl 2 (1 mM) or NiCl 2 (1 mM) using pUC19 (2xACCGG) containing the composite site 5′ CCGGT-N5-GGATCC-N5 ACCGG 3′. The duplex oligos containing the composite site was inserted into pUC19 and flanked by Hin dIII (AAGCTT) and Eco RI (GAATTC) sites. (B) Bottom-strand sequencing read (nicked top-strand template) near the Bam HI site. The composite site contains a nick as indicated by a strong A/C doublet. If the bottom strand is shown as a reverse-complement, a T/G doublet would be present (C) in the nicking site.
    Figure Legend Snippet: DNA run-off sequencing of the nicking sites and nicking distance from the Bam HI site. (A) Top-strand sequencing read from nicked bottom strand. Nicking reactions were carried out in NEB buffer 2 supplemented with MnCl 2 (1 mM) or NiCl 2 (1 mM) using pUC19 (2xACCGG) containing the composite site 5′ CCGGT-N5-GGATCC-N5 ACCGG 3′. The duplex oligos containing the composite site was inserted into pUC19 and flanked by Hin dIII (AAGCTT) and Eco RI (GAATTC) sites. (B) Bottom-strand sequencing read (nicked top-strand template) near the Bam HI site. The composite site contains a nick as indicated by a strong A/C doublet. If the bottom strand is shown as a reverse-complement, a T/G doublet would be present (C) in the nicking site.

    Techniques Used: Sequencing

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 96
    New England Biolabs haeiii
    The mutational effects in the laboratory drift correlate with sequence exchanges in <t>M.HaeIII</t> orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target <t>DNA</t> binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.
    Haeiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/haeiii/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    haeiii - by Bioz Stars, 2022-09
    96/100 stars
      Buy from Supplier

    95
    New England Biolabs protein kinase ckii buffer
    Enhanced binding of βarr1 to phosphorylated Dvl. ( A ) HEK-293 cells were transfected with Myc-Dvl2 and FLAG-βarr1, and cell extracts were immunoprecipitated with anti-Myc antibody directed against Dvl2. Immunoprecipitated Dvl2 was phosphorylated in the presence of [γ- 32 P]ATP by endogenous Dvl2-associated kinase(s). Immunoprecipitated Dvl2 was processed for autoradiography ( Upper ) or incubated with His-βarr1 for > 3 h at 4°C. Washed Dvl2 immunoprecipitates were analyzed by immunoblot with anti-His antibody to detect associated βarr1 ( Lower ). ( B ) <t>MBP-Dvl1</t> was phosphorylated by protein kinase <t>CKII</t> in the presence of ATP. Phosphorylated and unphosphorylated control MBP-Dvl1 was incubated with His-βarr1 for > 3 h at 4°C, and associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1. ( C ) MBP-Dvl1 was phosphorylated to various stoichiometric levels by protein kinase CKII. Phosphorylated MBP-Dvl1 was then either eluted in sample buffer and processed for autoradiography ( Inset Upper ) or incubated with His-βarr1. MBP-Dvl1-associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1 ( Inset Lower ). The relative amounts of βarr1 bound to MBP-Dvl1 phosphorylated by protein kinase CKII were quantified as fold increases over that bound to unphosphorylated MBP-Dvl1. Results are the mean ± SEM of three independent experiments.
    Protein Kinase Ckii Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein kinase ckii buffer/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protein kinase ckii buffer - by Bioz Stars, 2022-09
    95/100 stars
      Buy from Supplier

    97
    New England Biolabs non specific λ phosphatase
    Fig. 4. Enzymatic activities of immunoprecipitated NtCDPK2-myc. ( A ) Leaf discs of N . benthamiana that transiently expressed NtCDPK2-myc were harvested before (–) or after (+) infiltration of water. Solubilized membrane extracts were subjected to immunoprecipitation with monoclonal anti-c-myc antibodies, and precipitated proteins were analyzed by western blotting and immunodetected using a polyclonal c-myc antiserum. ( B ) Immunoprecipitates of NtCDPK2-myc before and after the flooding stimulus were prepared as in (A), and analyzed for CDPK autophosphorylation by incubation with [γ- 33 P]ATP (92 kBq) for the time indicated in the presence of 1 mM calcium or 5 mM EGTA (last data pair). Samples were separated on an SDS gel and analyzed by autoradiography. The two arrows indicate the non-elicited and elicited enzyme forms. ( C ) Leaf discs of Cf-9 tobacco that transiently expressed NtCDPK2-myc were harvested over a time course after elicitation with IF(Avr9 + ) or IF(Avr9 – ) (–). Solubilized membrane extracts were analyzed by western blotting (upper panel) and subjected to immunocomplex kinase assays with 100 µg/ml syntide-2 as substrate in the presence of 50 µM [γ- 33 P]ATP (92 kBq) and 1 mM calcium for 5 min at 30°C (lower panel). Supernatants werespotted on phosphocellulose paper and subjected to scintillation counting (phosphorylation of syntide-2; histogram). ( D ) NtCDPK2-myc was immunoprecipitated from N.benthamiana leaf extracts after a flooding stimulus as described in (A). Aliquots containing the immobilized enzyme were incubated with non-specific <t>λ</t> phosphatase for 10 min where indicated, or with phosphatase in the presence of 50 mM NaF and 10 mM Na 3 VO 4 (lane 3). After separating the phosphatase by washing, immunocomplex kinase assays were conducted as described in (C) with syntide-2 as substrate in the presence of 50 µM [γ- 33 P]ATP (92 kBq), 1 mM calcium (lanes 1–3 and 5) or 5 mM EGTA (lane 4). Supernatants were analyzed by western blotting (upper panel) as well as spotted on phosphocellulose paper and subjected to scintillation counting (lower panel).
    Non Specific λ Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/non specific λ phosphatase/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    non specific λ phosphatase - by Bioz Stars, 2022-09
    97/100 stars
      Buy from Supplier

    97
    New England Biolabs xho i restriction buffer
    Fig. 6. Xer recombination at psi and psiDC in the presence of XerC[De]. ( A ) Recombination of p- psi.psi and p- psiDC.psiDC in the presence of XerD and either XerC[De] or XerC. ( B ) Time course of recombination on p- psiDC.psiDC with XerC[De] and XerD. Reactions contained 40% glycerol and were cleaved with <t>Xho</t> I. Bands are indicated as follows: HJ, HJ intermediates; S, substrate fragments; P, product fragments.
    Xho I Restriction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xho i restriction buffer/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xho i restriction buffer - by Bioz Stars, 2022-09
    97/100 stars
      Buy from Supplier

    Image Search Results


    The mutational effects in the laboratory drift correlate with sequence exchanges in M.HaeIII orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target DNA binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.

    Journal: PLoS Computational Biology

    Article Title: Systematic Mapping of Protein Mutational Space by Prolonged Drift Reveals the Deleterious Effects of Seemingly Neutral Mutations

    doi: 10.1371/journal.pcbi.1004421

    Figure Lengend Snippet: The mutational effects in the laboratory drift correlate with sequence exchanges in M.HaeIII orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target DNA binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.

    Article Snippet: About 106 individual transformants were obtained in each round. (ii ) Colonies grown at 37°C overnight were combined, plasmid DNA was extracted and digested with HaeIII (10–20 units, in 50 μl of NEB buffer 2, for 2 hours at 37°C), and re-purified (PCR purification kit, QIAGEN). (iii ) The recovered plasmid DNA was re-transformed for another round of enrichment.

    Techniques: Sequencing, Functional Assay, Binding Assay

    Enhanced binding of βarr1 to phosphorylated Dvl. ( A ) HEK-293 cells were transfected with Myc-Dvl2 and FLAG-βarr1, and cell extracts were immunoprecipitated with anti-Myc antibody directed against Dvl2. Immunoprecipitated Dvl2 was phosphorylated in the presence of [γ- 32 P]ATP by endogenous Dvl2-associated kinase(s). Immunoprecipitated Dvl2 was processed for autoradiography ( Upper ) or incubated with His-βarr1 for > 3 h at 4°C. Washed Dvl2 immunoprecipitates were analyzed by immunoblot with anti-His antibody to detect associated βarr1 ( Lower ). ( B ) MBP-Dvl1 was phosphorylated by protein kinase CKII in the presence of ATP. Phosphorylated and unphosphorylated control MBP-Dvl1 was incubated with His-βarr1 for > 3 h at 4°C, and associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1. ( C ) MBP-Dvl1 was phosphorylated to various stoichiometric levels by protein kinase CKII. Phosphorylated MBP-Dvl1 was then either eluted in sample buffer and processed for autoradiography ( Inset Upper ) or incubated with His-βarr1. MBP-Dvl1-associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1 ( Inset Lower ). The relative amounts of βarr1 bound to MBP-Dvl1 phosphorylated by protein kinase CKII were quantified as fold increases over that bound to unphosphorylated MBP-Dvl1. Results are the mean ± SEM of three independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: ?-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins

    doi: 10.1073/pnas.211572798

    Figure Lengend Snippet: Enhanced binding of βarr1 to phosphorylated Dvl. ( A ) HEK-293 cells were transfected with Myc-Dvl2 and FLAG-βarr1, and cell extracts were immunoprecipitated with anti-Myc antibody directed against Dvl2. Immunoprecipitated Dvl2 was phosphorylated in the presence of [γ- 32 P]ATP by endogenous Dvl2-associated kinase(s). Immunoprecipitated Dvl2 was processed for autoradiography ( Upper ) or incubated with His-βarr1 for > 3 h at 4°C. Washed Dvl2 immunoprecipitates were analyzed by immunoblot with anti-His antibody to detect associated βarr1 ( Lower ). ( B ) MBP-Dvl1 was phosphorylated by protein kinase CKII in the presence of ATP. Phosphorylated and unphosphorylated control MBP-Dvl1 was incubated with His-βarr1 for > 3 h at 4°C, and associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1. ( C ) MBP-Dvl1 was phosphorylated to various stoichiometric levels by protein kinase CKII. Phosphorylated MBP-Dvl1 was then either eluted in sample buffer and processed for autoradiography ( Inset Upper ) or incubated with His-βarr1. MBP-Dvl1-associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1 ( Inset Lower ). The relative amounts of βarr1 bound to MBP-Dvl1 phosphorylated by protein kinase CKII were quantified as fold increases over that bound to unphosphorylated MBP-Dvl1. Results are the mean ± SEM of three independent experiments.

    Article Snippet: MBP-Dvl1 was washed with protein kinase CKII buffer (20 mM Tris, pH 7.5/50 mM KCl/2 mM MgCl2 ) and phosphorylated with protein kinase CKII (New England Biolabs) and [γ-32 P]ATP (60 μM; final concentration, 400 cpm/pmol) at 30°C for 30 min. Phosphorylated MBP-Dvl1 was visualized by exposure of dried gels to film.

    Techniques: Binding Assay, Transfection, Immunoprecipitation, Autoradiography, Incubation

    Fig. 4. Enzymatic activities of immunoprecipitated NtCDPK2-myc. ( A ) Leaf discs of N . benthamiana that transiently expressed NtCDPK2-myc were harvested before (–) or after (+) infiltration of water. Solubilized membrane extracts were subjected to immunoprecipitation with monoclonal anti-c-myc antibodies, and precipitated proteins were analyzed by western blotting and immunodetected using a polyclonal c-myc antiserum. ( B ) Immunoprecipitates of NtCDPK2-myc before and after the flooding stimulus were prepared as in (A), and analyzed for CDPK autophosphorylation by incubation with [γ- 33 P]ATP (92 kBq) for the time indicated in the presence of 1 mM calcium or 5 mM EGTA (last data pair). Samples were separated on an SDS gel and analyzed by autoradiography. The two arrows indicate the non-elicited and elicited enzyme forms. ( C ) Leaf discs of Cf-9 tobacco that transiently expressed NtCDPK2-myc were harvested over a time course after elicitation with IF(Avr9 + ) or IF(Avr9 – ) (–). Solubilized membrane extracts were analyzed by western blotting (upper panel) and subjected to immunocomplex kinase assays with 100 µg/ml syntide-2 as substrate in the presence of 50 µM [γ- 33 P]ATP (92 kBq) and 1 mM calcium for 5 min at 30°C (lower panel). Supernatants werespotted on phosphocellulose paper and subjected to scintillation counting (phosphorylation of syntide-2; histogram). ( D ) NtCDPK2-myc was immunoprecipitated from N.benthamiana leaf extracts after a flooding stimulus as described in (A). Aliquots containing the immobilized enzyme were incubated with non-specific λ phosphatase for 10 min where indicated, or with phosphatase in the presence of 50 mM NaF and 10 mM Na 3 VO 4 (lane 3). After separating the phosphatase by washing, immunocomplex kinase assays were conducted as described in (C) with syntide-2 as substrate in the presence of 50 µM [γ- 33 P]ATP (92 kBq), 1 mM calcium (lanes 1–3 and 5) or 5 mM EGTA (lane 4). Supernatants were analyzed by western blotting (upper panel) as well as spotted on phosphocellulose paper and subjected to scintillation counting (lower panel).

    Journal: The EMBO Journal

    Article Title: Calcium-dependent protein kinases play an essential role in a plant defence response

    doi: 10.1093/emboj/20.20.5556

    Figure Lengend Snippet: Fig. 4. Enzymatic activities of immunoprecipitated NtCDPK2-myc. ( A ) Leaf discs of N . benthamiana that transiently expressed NtCDPK2-myc were harvested before (–) or after (+) infiltration of water. Solubilized membrane extracts were subjected to immunoprecipitation with monoclonal anti-c-myc antibodies, and precipitated proteins were analyzed by western blotting and immunodetected using a polyclonal c-myc antiserum. ( B ) Immunoprecipitates of NtCDPK2-myc before and after the flooding stimulus were prepared as in (A), and analyzed for CDPK autophosphorylation by incubation with [γ- 33 P]ATP (92 kBq) for the time indicated in the presence of 1 mM calcium or 5 mM EGTA (last data pair). Samples were separated on an SDS gel and analyzed by autoradiography. The two arrows indicate the non-elicited and elicited enzyme forms. ( C ) Leaf discs of Cf-9 tobacco that transiently expressed NtCDPK2-myc were harvested over a time course after elicitation with IF(Avr9 + ) or IF(Avr9 – ) (–). Solubilized membrane extracts were analyzed by western blotting (upper panel) and subjected to immunocomplex kinase assays with 100 µg/ml syntide-2 as substrate in the presence of 50 µM [γ- 33 P]ATP (92 kBq) and 1 mM calcium for 5 min at 30°C (lower panel). Supernatants werespotted on phosphocellulose paper and subjected to scintillation counting (phosphorylation of syntide-2; histogram). ( D ) NtCDPK2-myc was immunoprecipitated from N.benthamiana leaf extracts after a flooding stimulus as described in (A). Aliquots containing the immobilized enzyme were incubated with non-specific λ phosphatase for 10 min where indicated, or with phosphatase in the presence of 50 mM NaF and 10 mM Na 3 VO 4 (lane 3). After separating the phosphatase by washing, immunocomplex kinase assays were conducted as described in (C) with syntide-2 as substrate in the presence of 50 µM [γ- 33 P]ATP (92 kBq), 1 mM calcium (lanes 1–3 and 5) or 5 mM EGTA (lane 4). Supernatants were analyzed by western blotting (upper panel) as well as spotted on phosphocellulose paper and subjected to scintillation counting (lower panel).

    Article Snippet: For in vitro interconversion experiments, reactions with NtCDPK2-myc immunoprecipitates/beads were washed once with phosphatase buffer (NEB) and resuspended in 100 µl of the same buffer containing 2 U of non-specific λ phosphatase (NEB) in the absence or presence (control) of 50 mM NaF and 10 mM Na3 VO4 .

    Techniques: Immunoprecipitation, Western Blot, Incubation, SDS-Gel, Autoradiography

    Fig. 6. Xer recombination at psi and psiDC in the presence of XerC[De]. ( A ) Recombination of p- psi.psi and p- psiDC.psiDC in the presence of XerD and either XerC[De] or XerC. ( B ) Time course of recombination on p- psiDC.psiDC with XerC[De] and XerD. Reactions contained 40% glycerol and were cleaved with Xho I. Bands are indicated as follows: HJ, HJ intermediates; S, substrate fragments; P, product fragments.

    Journal: The EMBO Journal

    Article Title: Accessory factors determine the order of strand exchange in Xer recombination at psi

    doi: 10.1093/emboj/cdf379

    Figure Lengend Snippet: Fig. 6. Xer recombination at psi and psiDC in the presence of XerC[De]. ( A ) Recombination of p- psi.psi and p- psiDC.psiDC in the presence of XerD and either XerC[De] or XerC. ( B ) Time course of recombination on p- psiDC.psiDC with XerC[De] and XerD. Reactions contained 40% glycerol and were cleaved with Xho I. Bands are indicated as follows: HJ, HJ intermediates; S, substrate fragments; P, product fragments.

    Article Snippet: The other lane was excised from the gel and equilibrated for 2 h with two changes in Xho I restriction buffer (New England Biolabs NEBuffer 2 + BSA).

    Techniques: