anti mbp magnetic beads  (New England Biolabs)


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    Name:
    Anti MBP Magnetic Beads
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    Anti MBP Magnetic Beads 10 mg
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    e8037s
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    10 mg
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    Protein Purification Kit Components
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    New England Biolabs anti mbp magnetic beads
    Anti MBP Magnetic Beads
    Anti MBP Magnetic Beads 10 mg
    https://www.bioz.com/result/anti mbp magnetic beads/product/New England Biolabs
    Average 95 stars, based on 67 article reviews
    Price from $9.99 to $1999.99
    anti mbp magnetic beads - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria"

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria

    Journal: mBio

    doi: 10.1128/mBio.00809-18

    MrrA phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the MBP-CC2874 preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.
    Figure Legend Snippet: MrrA phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the MBP-CC2874 preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.

    Techniques Used: SDS Page, Autoradiography, Activity Assay, Purification

    LovK and PhyK are phosphotransferases that are activated by MrrA~P. (A) Schematic representation of two possible modes of action of MrrA~P, phosphotransfer to and allosteric activation of PhyK and LovK. For reasons of simplicity, only PhyK is shown. (B) PhyK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, the kinase CC2874, MrrA, and different PhyK variants. Purified PhyK wild type or mutant variants harboring mutations in the G1 (G514A/G516A) or G2 (G526A) box of the ATP-binding site were used as indicated. The positions of phosphorylated proteins on the gel are indicated on the right. (C) LovK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, kinase CC2874, MrrA, and different LovK variants. Purified LovK wild type or mutant variants harboring mutations in the G1 (G319A/G321A) or G2 (G332A) box ATP-binding site of the CA domain were used as indicated. MrrA dilution factors are indicated in each lane. The positions of phosphorylated proteins on the gel are indicated on the right. (D) Phosphotransfer to PhyR does not require a conserved CA domain. Phosphorylation reaction mixtures containing radiolabeled ATP, kinase CC2874, MrrA, PhyR, NepR, and different PhyK variants are as in panel C. The positions of phosphorylated proteins on the gel are indicated on the right. (E) Preparation of purified MrrA~P. Kinase CC2874 alone or with MrrA was phosphorylated with radiolabeled ATP (lanes 1 and 7), and CC2874 was subsequently removed using anti-MBP magnetic beads (lanes 2 and 8). Next, ATP was hydrolyzed by treating mixtures with hexokinase and glucose (lanes 3 and 9). CC2874 was added back to ATP-depleted samples, and mixtures were incubated for 0.5, 1.0, and 5 min (lanes 4 to 6 and 10 to 12). (F) Purified MrrA~P transfers phosphate to LovK. MrrA~P was prepared as in panel E (lane 1), CC2874 was removed (lane 2), and ATP was degraded (lane 3). Fresh CC2874 (lane 4) or LovK was added, and phosphotransfer from MrrA~P was monitored after 10 s, 20 s, 1 min, 2 min, 10 min, and 20 min (lanes 5 to 10, respectively). (G) ATP binding is not required for PhyK activity in the general stress response. Δ lovK strains harboring an empty vector (EV) or a plasmid expressing different phyK alleles from a cumate-inducible promoter were analyzed. Plasmid-driven variants of PhyK contained mutations in the G1 or G2 box of the ATP-binding pocket (see above) or in the conserved phosphoacceptor His371. SigT-dependent sigU promoter activity (Miller units) was determined using a lacZ promoter fusion in strains grown in the presence (+) or absence (−) of cumate. PhyK variants harbored a C-terminal 3×FLAG tag that allowed monitoring their expression by immunoblot analysis (lower panels). An immunoblot with anti-MreB antibodies is shown as a control. Note that the sigUp-lacZ reporter fusion used in these experiments differed from the one used in experiments above ( Fig. 1D ) and shows higher basal activity (compare wild-type PhyK and empty-vector control in panel G).
    Figure Legend Snippet: LovK and PhyK are phosphotransferases that are activated by MrrA~P. (A) Schematic representation of two possible modes of action of MrrA~P, phosphotransfer to and allosteric activation of PhyK and LovK. For reasons of simplicity, only PhyK is shown. (B) PhyK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, the kinase CC2874, MrrA, and different PhyK variants. Purified PhyK wild type or mutant variants harboring mutations in the G1 (G514A/G516A) or G2 (G526A) box of the ATP-binding site were used as indicated. The positions of phosphorylated proteins on the gel are indicated on the right. (C) LovK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, kinase CC2874, MrrA, and different LovK variants. Purified LovK wild type or mutant variants harboring mutations in the G1 (G319A/G321A) or G2 (G332A) box ATP-binding site of the CA domain were used as indicated. MrrA dilution factors are indicated in each lane. The positions of phosphorylated proteins on the gel are indicated on the right. (D) Phosphotransfer to PhyR does not require a conserved CA domain. Phosphorylation reaction mixtures containing radiolabeled ATP, kinase CC2874, MrrA, PhyR, NepR, and different PhyK variants are as in panel C. The positions of phosphorylated proteins on the gel are indicated on the right. (E) Preparation of purified MrrA~P. Kinase CC2874 alone or with MrrA was phosphorylated with radiolabeled ATP (lanes 1 and 7), and CC2874 was subsequently removed using anti-MBP magnetic beads (lanes 2 and 8). Next, ATP was hydrolyzed by treating mixtures with hexokinase and glucose (lanes 3 and 9). CC2874 was added back to ATP-depleted samples, and mixtures were incubated for 0.5, 1.0, and 5 min (lanes 4 to 6 and 10 to 12). (F) Purified MrrA~P transfers phosphate to LovK. MrrA~P was prepared as in panel E (lane 1), CC2874 was removed (lane 2), and ATP was degraded (lane 3). Fresh CC2874 (lane 4) or LovK was added, and phosphotransfer from MrrA~P was monitored after 10 s, 20 s, 1 min, 2 min, 10 min, and 20 min (lanes 5 to 10, respectively). (G) ATP binding is not required for PhyK activity in the general stress response. Δ lovK strains harboring an empty vector (EV) or a plasmid expressing different phyK alleles from a cumate-inducible promoter were analyzed. Plasmid-driven variants of PhyK contained mutations in the G1 or G2 box of the ATP-binding pocket (see above) or in the conserved phosphoacceptor His371. SigT-dependent sigU promoter activity (Miller units) was determined using a lacZ promoter fusion in strains grown in the presence (+) or absence (−) of cumate. PhyK variants harbored a C-terminal 3×FLAG tag that allowed monitoring their expression by immunoblot analysis (lower panels). An immunoblot with anti-MreB antibodies is shown as a control. Note that the sigUp-lacZ reporter fusion used in these experiments differed from the one used in experiments above ( Fig. 1D ) and shows higher basal activity (compare wild-type PhyK and empty-vector control in panel G).

    Techniques Used: Activation Assay, Purification, Mutagenesis, Binding Assay, Magnetic Beads, Incubation, Activity Assay, Plasmid Preparation, Expressing

    2) Product Images from "Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2"

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023668

    SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.
    Figure Legend Snippet: SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Sequencing, Construct, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

    SIRV2 Hjr interacts with SIRV2gp26 coat protein in vitro . The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.
    Figure Legend Snippet: SIRV2 Hjr interacts with SIRV2gp26 coat protein in vitro . The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.

    Techniques Used: In Vitro, Immunoprecipitation, SDS Page, Staining

    3) Product Images from "The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201"

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01584

    ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P
    Figure Legend Snippet: ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, In Vivo, Polymerase Chain Reaction, Generated, Immunoprecipitation, Isolation, Magnetic Beads, Quantitative RT-PCR, Derivative Assay

    4) Product Images from "The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201"

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01584

    ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P
    Figure Legend Snippet: ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, In Vivo, Polymerase Chain Reaction, Generated, Immunoprecipitation, Isolation, Magnetic Beads, Quantitative RT-PCR, Derivative Assay

    5) Product Images from "Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2"

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023668

    SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.
    Figure Legend Snippet: SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Sequencing, Construct, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

    SIRV2 Hjr interacts with SIRV2gp26 coat protein in vitro . The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.
    Figure Legend Snippet: SIRV2 Hjr interacts with SIRV2gp26 coat protein in vitro . The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.

    Techniques Used: In Vitro, Immunoprecipitation, SDS Page, Staining

    6) Product Images from "A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria"

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria

    Journal: mBio

    doi: 10.1128/mBio.00809-18

    MrrA phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the MBP-CC2874 preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.
    Figure Legend Snippet: MrrA phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the MBP-CC2874 preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.

    Techniques Used: SDS Page, Autoradiography, Activity Assay, Purification

    Related Articles

    Centrifugation:

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201
    Article Snippet: After the removal of cell debris by centrifugation, we used 50 μL of each sample as an input control. .. The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples.

    Construct:

    Article Title: A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control
    Article Snippet: The MBP-DST plasmid was constructed by digesting the pDST plasmid with HindIII and SalI to release the ORF of DST , which was then inserted into the HindIII–SalI site of pMAL-c2X (New England Biolabs). .. The MBP-DST fusion protein was prepared as described above and purified with anti-MBP magnetic beads (New England Biolabs) according to the manufacturer's instructions.

    SDS-Gel:

    Article Title: Understanding the role of the Q338H MUTYH variant in oxidative damage repair
    Article Snippet: The samples were then added to 30 µl of anti-MBP magnetic beads (1 mg/ml and binding capacity of 10 µg/mg) (New England BioLabs, Ipswich, MA, USA), pre-treated as described by manufacturer. .. The bead pellets were resuspended in 40 µl of Laemnli buffer 3 × [187.5 mM Tris–HCl, pH 6.8, 6% (w/v) SDS, 30% glycerol, 150 mM DTT and 0.03% (w/v) bromophenol blue] and heated at 70°C for 5 min. Supernatants (20 µl) were loaded on an SDS–gel NuPAGE 4–12% (Novex, Invitrogen), and proteins were transferred to a nitrocellulose membrane for western blot analysis.

    Incubation:

    Article Title: Understanding the role of the Q338H MUTYH variant in oxidative damage repair
    Article Snippet: Wild-type and variant MBP–MUTYH proteins (5 µg) were incubated with HeLa nuclear extracts overnight at 4°C in HEPES–KOH 10 mM, pH 7.4, KCl 100 mM and MgCl2 10 mM buffer. .. The samples were then added to 30 µl of anti-MBP magnetic beads (1 mg/ml and binding capacity of 10 µg/mg) (New England BioLabs, Ipswich, MA, USA), pre-treated as described by manufacturer.

    Article Title: A conserved membrane-binding domain targets proteins to organelle contact sites
    Article Snippet: Liposomes with various lipid composition and a trace amount of [3 H]triolein (American Radiolabled Chemicals) were added to the magnetic beads to a final concentration of 1 mM in a total volume 100 μl and incubated at 30°C for 30 minutes. .. The amount of MBP fusion protein was determined by immunoblotting with anti-MBP antibody (New England Biolabs) and comparing the results with those of known amounts of pure MBP.

    Article Title: Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1
    Article Snippet: His-MBP-tagged UHRF1 (1 µM) was incubated with conjugated beads in pulldown buffer (200 µL) overnight at 4 °C in the presence of increasing concentrations (0.5, 1, 10, 50, and 100 µM) of HeDNA (same oligonucleotide as in ubiquitination reaction). .. For Western blot, 10 µL of bound protein and 2% of the unbound fraction was loaded for input onto SDS/PAGE.), 5 pmol of MBP-DNMT1 was bound to 5 µL of anti-MBP magnetic beads (New England BioLabs), and the remainder of the procedure was identical to the previously described pulldown.

    Article Title: DNA-PK-mediated phosphorylation of EZH2 regulates the DNA damage-induced apoptosis to maintain T-cell genomic integrity
    Article Snippet: .. MBP-tagged proteins were captured by incubation with anti-MBP magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 3 h. The bead pellet was washed five times with 500 μ l of buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 10% glycerol and 2 mM EDTA). ..

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria
    Article Snippet: .. CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet. .. Hexokinase and glucose were added to the supernatant as described above and incubated for 10 min to deplete remaining ATP.

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201
    Article Snippet: The culture was incubated with shaking at 28°C for 15 min. To quench the cross-linking reaction, glycine was added to make a final concentration of 250 mM, followed by a 15 min incubation at room temperature. .. The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples.

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.). .. After a 1-h incubation a 4°C, immunoprecipitates were washed five times in PBS containing 0.1% Triton X-100 and an additional 250 mM NaCl.

    Article Title: Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1
    Article Snippet: Next, oMBP-DNMT1 (0.5 µM) was incubated with the bound nucleosomes overnight at 4 °C in pulldown buffer (200 µL). .. For the pulldown of ubiquitinated nucleosomes by DNMT1 (SI Appendix , Fig. S3D ), 5 pmol of MBP-DNMT1 was bound to 5 µL of anti-MBP magnetic beads (New England BioLabs), and the remainder of the procedure was identical to the previously described pulldown.

    Article Title: DCA1 Acts as a Transcriptional Co-activator of DST and Contributes to Drought and Salt Tolerance in Rice
    Article Snippet: .. The supernatant was incubated with Anti-MBP Magnetic Beads (NEB) for 30 min at 4°C and washed five times with MBP column buffer. .. Then, supernatants containing DCA1-His2 and His2 proteins were incubated with approximately the same amount of MBP and DST-MBP binding beads for 2 h at 4°C.

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2
    Article Snippet: In a 0.5 mL reaction, 10 µg MBP-Hjr and incubated with ∼1 mg SIRV2-infected S. islandicus extract at 4°C for 16 hours with shaking in NEBuffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.9 @ 25°C). .. Anti-MBP magnetic beads were added and affinity complexes were magnetically separated and washed five times with 1.0 mL of NEBuffer 3 to elute non-specifically bound protein.

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2
    Article Snippet: Interaction between Hjr and SIRV2gp26 coat protein To directly test the interaction between Hjr and SIRV2gp26 coat protein in vitro , anti-MBP::MBP-Hjr magnetic beads (hereafter called MBP-Hjr affinity beads) were prepared by mixing 10 µg MBP-Hjr with 0.1 mg anti-MBP magnetic beads pre-equilibrated in NEBuffer 3 (New England Biolabs). .. Bead complexes were mixed thoroughly and incubated at 4°C with shaking for 1 hour.

    Activity Assay:

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria
    Article Snippet: In experiments assessing phosphatase activity, ATP was depleted by the addition of 1.5 units of hexokinase (Roche) and 5 mM d -glucose 15 min after phosphorylation. .. CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet.

    Expressing:

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.). ..

    Article Title: DCA1 Acts as a Transcriptional Co-activator of DST and Contributes to Drought and Salt Tolerance in Rice
    Article Snippet: Briefly, E . coli cells expressing DST-MBP and MBP were lysed with BugBuster Protein Extraction Reagents (Novagen) and centrifuged. .. The supernatant was incubated with Anti-MBP Magnetic Beads (NEB) for 30 min at 4°C and washed five times with MBP column buffer.

    Article Title: A conserved membrane-binding domain targets proteins to organelle contact sites
    Article Snippet: Wild-type yeast cells expressing MBP–SMP fusions were grown to mid-logarithmic phase and lysed by agitation with glass beads in Mini-BeadBeater-8 (Biospec Products). .. The cell lysates (from four OD600nm units of culture) were mixed with magnetic beads coated with 400 μg anti-MBP antibody (New England Biolabs) for 1 hour on ice and mixed every 15 minutes.

    Modification:

    Article Title: Understanding the role of the Q338H MUTYH variant in oxidative damage repair
    Article Snippet: Pull-down assays HeLa cells were grown in low glucose Dulbecco’s Modified Essential Medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin and 100 µg/ml of streptomycin (complete medium) at 37°C in 5% CO2 atmosphere. .. The samples were then added to 30 µl of anti-MBP magnetic beads (1 mg/ml and binding capacity of 10 µg/mg) (New England BioLabs, Ipswich, MA, USA), pre-treated as described by manufacturer.

    Western Blot:

    Article Title: Understanding the role of the Q338H MUTYH variant in oxidative damage repair
    Article Snippet: The samples were then added to 30 µl of anti-MBP magnetic beads (1 mg/ml and binding capacity of 10 µg/mg) (New England BioLabs, Ipswich, MA, USA), pre-treated as described by manufacturer. .. The bead pellets were resuspended in 40 µl of Laemnli buffer 3 × [187.5 mM Tris–HCl, pH 6.8, 6% (w/v) SDS, 30% glycerol, 150 mM DTT and 0.03% (w/v) bromophenol blue] and heated at 70°C for 5 min. Supernatants (20 µl) were loaded on an SDS–gel NuPAGE 4–12% (Novex, Invitrogen), and proteins were transferred to a nitrocellulose membrane for western blot analysis.

    Article Title: Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1
    Article Snippet: .. For Western blot, 10 µL of bound protein and 2% of the unbound fraction was loaded for input onto SDS/PAGE.), 5 pmol of MBP-DNMT1 was bound to 5 µL of anti-MBP magnetic beads (New England BioLabs), and the remainder of the procedure was identical to the previously described pulldown. .. Proteins were separated by SDS/PAGE and transferred to PVDF membrane (Amersham Hybond P), using a semidry transfer apparatus (Hoefer) for 1.5 h at a current of 1 mA/cm2 .

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.). ..

    Transformation Assay:

    Article Title: A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control
    Article Snippet: The transformed cells were cultured at 37°C until the OD600 of the cell culture was 0.5, and then induced with 1 mM IPTG (isopropyl-1-thio-β-D-galactopyranoside) for 36 h at 12°C. .. The MBP-DST fusion protein was prepared as described above and purified with anti-MBP magnetic beads (New England Biolabs) according to the manufacturer's instructions.

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: The Mps3 SUN domain was also purified from BL21(DE3) pLysS bacteria transformed with pQE10-mps3SUN (pSJ413) by metal affinity chromatography, and 0.5 mg purified 6xHis-mps3SUN labeled with Alexa 680 as described previously ( ). .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.).

    Immunoprecipitation:

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201
    Article Snippet: .. The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples. .. After incubation at 4°C overnight, beads were pelleted and washed three times with 0.1 M NaPhosphate buffer (pH 8.0).

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.). .. 100 μl of each extract was mixed with 1 μg of purified 6xHis-mps3SUN in a 500-μl immunoprecipitation that included 2 μl protein A–coated magnetic beads (Dynal) and 1 μl of affinity-purified anti-Mps2 antibodies.

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2
    Article Snippet: Paragraph title: Immunoprecipitation ... Anti-MBP magnetic beads were added and affinity complexes were magnetically separated and washed five times with 1.0 mL of NEBuffer 3 to elute non-specifically bound protein.

    Cell Culture:

    Article Title: A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control
    Article Snippet: For extraction of native fusion protein, the cultured bacteria cells were lysed by using a high-pressure cell crusher and the fusion proteins were purified with Ni-NTA resin (Qiagen). .. The MBP-DST fusion protein was prepared as described above and purified with anti-MBP magnetic beads (New England Biolabs) according to the manufacturer's instructions.

    Generated:

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: Mps2 antibodies were generated by injecting a KLH-coupled peptide corresponding to residues 212–233 of Mps2 into guinea pigs (Pocono) followed by affinity purification on MBP-Mps2 bound to nitrocellulose filters. .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.).

    Imaging:

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria
    Article Snippet: Wet gels were exposed to phosphor screens (0.5 to 1.5 h) before being scanned using a Typhoon FLA7000 imaging system (GE Healthcare). .. CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet.

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: Membranes were probed with 10 μg Alexa 680–labeled 6xHis-mps3SUN, and binding was assessed using the Odyssey imaging system (LiCor). .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.).

    Sonication:

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201
    Article Snippet: Chromosomal DNA was sheared by sonication to an average size of 2–5 Kb, following the manufacturer’s instructions (Sonics Uibracell, United States) as output watts of 20 kW for 15 min, amplitude of 40% and pulse durations of 3 s ON and 6 s OFF. .. The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples.

    Affinity Purification:

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: Mps2 antibodies were generated by injecting a KLH-coupled peptide corresponding to residues 212–233 of Mps2 into guinea pigs (Pocono) followed by affinity purification on MBP-Mps2 bound to nitrocellulose filters. .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.).

    Binding Assay:

    Article Title: Understanding the role of the Q338H MUTYH variant in oxidative damage repair
    Article Snippet: .. The samples were then added to 30 µl of anti-MBP magnetic beads (1 mg/ml and binding capacity of 10 µg/mg) (New England BioLabs, Ipswich, MA, USA), pre-treated as described by manufacturer. .. The bead pellets were resuspended in 40 µl of Laemnli buffer 3 × [187.5 mM Tris–HCl, pH 6.8, 6% (w/v) SDS, 30% glycerol, 150 mM DTT and 0.03% (w/v) bromophenol blue] and heated at 70°C for 5 min. Supernatants (20 µl) were loaded on an SDS–gel NuPAGE 4–12% (Novex, Invitrogen), and proteins were transferred to a nitrocellulose membrane for western blot analysis.

    Article Title: A conserved membrane-binding domain targets proteins to organelle contact sites
    Article Snippet: Paragraph title: Liposome binding assay ... The amount of MBP fusion protein was determined by immunoblotting with anti-MBP antibody (New England Biolabs) and comparing the results with those of known amounts of pure MBP.

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: Membranes were probed with 10 μg Alexa 680–labeled 6xHis-mps3SUN, and binding was assessed using the Odyssey imaging system (LiCor). .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.).

    Article Title: DCA1 Acts as a Transcriptional Co-activator of DST and Contributes to Drought and Salt Tolerance in Rice
    Article Snippet: The supernatant was incubated with Anti-MBP Magnetic Beads (NEB) for 30 min at 4°C and washed five times with MBP column buffer. .. Then, supernatants containing DCA1-His2 and His2 proteins were incubated with approximately the same amount of MBP and DST-MBP binding beads for 2 h at 4°C.

    Pull Down Assay:

    Article Title: DNA-PK-mediated phosphorylation of EZH2 regulates the DNA damage-induced apoptosis to maintain T-cell genomic integrity
    Article Snippet: Paragraph title: MBP-EZH2 and GST-Ku80 protein purification and pull-down assay ... MBP-tagged proteins were captured by incubation with anti-MBP magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 3 h. The bead pellet was washed five times with 500 μ l of buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 10% glycerol and 2 mM EDTA).

    Article Title: DCA1 Acts as a Transcriptional Co-activator of DST and Contributes to Drought and Salt Tolerance in Rice
    Article Snippet: Paragraph title: Pull-Down Assay ... The supernatant was incubated with Anti-MBP Magnetic Beads (NEB) for 30 min at 4°C and washed five times with MBP column buffer.

    Magnetic Beads:

    Article Title: Understanding the role of the Q338H MUTYH variant in oxidative damage repair
    Article Snippet: .. The samples were then added to 30 µl of anti-MBP magnetic beads (1 mg/ml and binding capacity of 10 µg/mg) (New England BioLabs, Ipswich, MA, USA), pre-treated as described by manufacturer. .. The bead pellets were resuspended in 40 µl of Laemnli buffer 3 × [187.5 mM Tris–HCl, pH 6.8, 6% (w/v) SDS, 30% glycerol, 150 mM DTT and 0.03% (w/v) bromophenol blue] and heated at 70°C for 5 min. Supernatants (20 µl) were loaded on an SDS–gel NuPAGE 4–12% (Novex, Invitrogen), and proteins were transferred to a nitrocellulose membrane for western blot analysis.

    Article Title: A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control
    Article Snippet: .. The MBP-DST fusion protein was prepared as described above and purified with anti-MBP magnetic beads (New England Biolabs) according to the manufacturer's instructions. .. To generate the 6xHis-PRX, the ORF of peroxidase 24 precursor was PCR-amplified with the primers listed in Supplemental Table S2.

    Article Title: A conserved membrane-binding domain targets proteins to organelle contact sites
    Article Snippet: Liposomes with various lipid composition and a trace amount of [3 H]triolein (American Radiolabled Chemicals) were added to the magnetic beads to a final concentration of 1 mM in a total volume 100 μl and incubated at 30°C for 30 minutes. .. The amount of MBP fusion protein was determined by immunoblotting with anti-MBP antibody (New England Biolabs) and comparing the results with those of known amounts of pure MBP.

    Article Title: Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1
    Article Snippet: .. For Western blot, 10 µL of bound protein and 2% of the unbound fraction was loaded for input onto SDS/PAGE.), 5 pmol of MBP-DNMT1 was bound to 5 µL of anti-MBP magnetic beads (New England BioLabs), and the remainder of the procedure was identical to the previously described pulldown. .. Proteins were separated by SDS/PAGE and transferred to PVDF membrane (Amersham Hybond P), using a semidry transfer apparatus (Hoefer) for 1.5 h at a current of 1 mA/cm2 .

    Article Title: DNA-PK-mediated phosphorylation of EZH2 regulates the DNA damage-induced apoptosis to maintain T-cell genomic integrity
    Article Snippet: .. MBP-tagged proteins were captured by incubation with anti-MBP magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 3 h. The bead pellet was washed five times with 500 μ l of buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 10% glycerol and 2 mM EDTA). ..

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria
    Article Snippet: .. CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet. .. Hexokinase and glucose were added to the supernatant as described above and incubated for 10 min to deplete remaining ATP.

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201
    Article Snippet: .. The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples. .. After incubation at 4°C overnight, beads were pelleted and washed three times with 0.1 M NaPhosphate buffer (pH 8.0).

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.). .. 100 μl of each extract was mixed with 1 μg of purified 6xHis-mps3SUN in a 500-μl immunoprecipitation that included 2 μl protein A–coated magnetic beads (Dynal) and 1 μl of affinity-purified anti-Mps2 antibodies.

    Article Title: Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1
    Article Snippet: .. For the pulldown of ubiquitinated nucleosomes by DNMT1 (SI Appendix , Fig. S3D ), 5 pmol of MBP-DNMT1 was bound to 5 µL of anti-MBP magnetic beads (New England BioLabs), and the remainder of the procedure was identical to the previously described pulldown. .. Proteins were separated by SDS/PAGE and transferred to PVDF membrane (Amersham Hybond P), using a semidry transfer apparatus (Hoefer) for 1.5 h at a current of 1 mA/cm2 .

    Article Title: DCA1 Acts as a Transcriptional Co-activator of DST and Contributes to Drought and Salt Tolerance in Rice
    Article Snippet: .. The supernatant was incubated with Anti-MBP Magnetic Beads (NEB) for 30 min at 4°C and washed five times with MBP column buffer. .. Then, supernatants containing DCA1-His2 and His2 proteins were incubated with approximately the same amount of MBP and DST-MBP binding beads for 2 h at 4°C.

    Article Title: A conserved membrane-binding domain targets proteins to organelle contact sites
    Article Snippet: .. The cell lysates (from four OD600nm units of culture) were mixed with magnetic beads coated with 400 μg anti-MBP antibody (New England Biolabs) for 1 hour on ice and mixed every 15 minutes. ..

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2
    Article Snippet: .. Anti-MBP magnetic beads were added and affinity complexes were magnetically separated and washed five times with 1.0 mL of NEBuffer 3 to elute non-specifically bound protein. .. The remaining specific protein complexes were eluted by boiling in 1× SDS-PAGE loading buffer for 5 minutes and analyzed by SDS-PAGE.

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2
    Article Snippet: .. Interaction between Hjr and SIRV2gp26 coat protein To directly test the interaction between Hjr and SIRV2gp26 coat protein in vitro , anti-MBP::MBP-Hjr magnetic beads (hereafter called MBP-Hjr affinity beads) were prepared by mixing 10 µg MBP-Hjr with 0.1 mg anti-MBP magnetic beads pre-equilibrated in NEBuffer 3 (New England Biolabs). .. Bead complexes were mixed thoroughly and incubated at 4°C with shaking for 1 hour.

    Labeling:

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: The Mps3 SUN domain was also purified from BL21(DE3) pLysS bacteria transformed with pQE10-mps3SUN (pSJ413) by metal affinity chromatography, and 0.5 mg purified 6xHis-mps3SUN labeled with Alexa 680 as described previously ( ). .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.).

    Purification:

    Article Title: A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control
    Article Snippet: .. The MBP-DST fusion protein was prepared as described above and purified with anti-MBP magnetic beads (New England Biolabs) according to the manufacturer's instructions. .. To generate the 6xHis-PRX, the ORF of peroxidase 24 precursor was PCR-amplified with the primers listed in Supplemental Table S2.

    Article Title: DNA-PK-mediated phosphorylation of EZH2 regulates the DNA damage-induced apoptosis to maintain T-cell genomic integrity
    Article Snippet: Purified protein was eluted with elution buffer (500 mM imidazole, 1 M NaCl, 10% glycerol, and 50 mM Tris-HCl (pH 8.0)). .. MBP-tagged proteins were captured by incubation with anti-MBP magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 3 h. The bead pellet was washed five times with 500 μ l of buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 10% glycerol and 2 mM EDTA).

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria
    Article Snippet: For the purification of MrrA~P, the following conditions were used. .. CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet.

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: The Mps3 SUN domain was also purified from BL21(DE3) pLysS bacteria transformed with pQE10-mps3SUN (pSJ413) by metal affinity chromatography, and 0.5 mg purified 6xHis-mps3SUN labeled with Alexa 680 as described previously ( ). .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.).

    Protein Purification:

    Article Title: DNA-PK-mediated phosphorylation of EZH2 regulates the DNA damage-induced apoptosis to maintain T-cell genomic integrity
    Article Snippet: Paragraph title: MBP-EZH2 and GST-Ku80 protein purification and pull-down assay ... MBP-tagged proteins were captured by incubation with anti-MBP magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 3 h. The bead pellet was washed five times with 500 μ l of buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 10% glycerol and 2 mM EDTA).

    Protein Extraction:

    Article Title: DCA1 Acts as a Transcriptional Co-activator of DST and Contributes to Drought and Salt Tolerance in Rice
    Article Snippet: Briefly, E . coli cells expressing DST-MBP and MBP were lysed with BugBuster Protein Extraction Reagents (Novagen) and centrifuged. .. The supernatant was incubated with Anti-MBP Magnetic Beads (NEB) for 30 min at 4°C and washed five times with MBP column buffer.

    Lysis:

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201
    Article Snippet: Cell pellets were collected and washed twice with 1 × PBS buffer and were then re-suspended in lysis buffer (20 mM Tris–HCl, 50 mM KCl, 0.5 mM DTT, 10% glycerol, and 5 mM protein inhibitor, pH 7.9). .. The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples.

    Chromatin Immunoprecipitation:

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation (ChIP) Assay ... The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples.

    SDS Page:

    Article Title: A conserved membrane-binding domain targets proteins to organelle contact sites
    Article Snippet: The beads were mixed with 2× SDS sample buffer and proteins bound were separated by SDS-PAGE. .. The amount of MBP fusion protein was determined by immunoblotting with anti-MBP antibody (New England Biolabs) and comparing the results with those of known amounts of pure MBP.

    Article Title: Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1
    Article Snippet: .. For Western blot, 10 µL of bound protein and 2% of the unbound fraction was loaded for input onto SDS/PAGE.), 5 pmol of MBP-DNMT1 was bound to 5 µL of anti-MBP magnetic beads (New England BioLabs), and the remainder of the procedure was identical to the previously described pulldown. .. Proteins were separated by SDS/PAGE and transferred to PVDF membrane (Amersham Hybond P), using a semidry transfer apparatus (Hoefer) for 1.5 h at a current of 1 mA/cm2 .

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: Cells from 1 ml of culture were resuspended in 200 μl 2× SDS sample buffer and heated to 100°C for 5 min, 5 μl of MBP-Mps2 and MBP-Kar1 and 2 μl of MBP-only samples were separated by 8% SDS-PAGE, and proteins were electrophoretically transferred to nitrocellulose. .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.).

    Article Title: DCA1 Acts as a Transcriptional Co-activator of DST and Contributes to Drought and Salt Tolerance in Rice
    Article Snippet: The supernatant was incubated with Anti-MBP Magnetic Beads (NEB) for 30 min at 4°C and washed five times with MBP column buffer. .. The mixture was resuspended in SDS loading buffer, boiled for 3 min, separated by 10% SDS-PAGE and immunoblotted with anti-MBP antibody for target proteins and anti-His antibody for pull-down proteins (CWbiotech).

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2
    Article Snippet: Anti-MBP magnetic beads were added and affinity complexes were magnetically separated and washed five times with 1.0 mL of NEBuffer 3 to elute non-specifically bound protein. .. The remaining specific protein complexes were eluted by boiling in 1× SDS-PAGE loading buffer for 5 minutes and analyzed by SDS-PAGE.

    Plasmid Preparation:

    Article Title: A previously unknown zinc finger protein, DST, regulates drought and salt tolerance in rice via stomatal aperture control
    Article Snippet: The MBP-DST plasmid was constructed by digesting the pDST plasmid with HindIII and SalI to release the ORF of DST , which was then inserted into the HindIII–SalI site of pMAL-c2X (New England Biolabs). .. The MBP-DST fusion protein was prepared as described above and purified with anti-MBP magnetic beads (New England Biolabs) according to the manufacturer's instructions.

    Affinity Chromatography:

    Article Title: The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope
    Article Snippet: The Mps3 SUN domain was also purified from BL21(DE3) pLysS bacteria transformed with pQE10-mps3SUN (pSJ413) by metal affinity chromatography, and 0.5 mg purified 6xHis-mps3SUN labeled with Alexa 680 as described previously ( ). .. Expression of bacterial proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-MBP antibodies (New England Biolabs, Inc.).

    In Vitro:

    Article Title: Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1
    Article Snippet: Paragraph title: In Vitro Pulldowns. ... For Western blot, 10 µL of bound protein and 2% of the unbound fraction was loaded for input onto SDS/PAGE.), 5 pmol of MBP-DNMT1 was bound to 5 µL of anti-MBP magnetic beads (New England BioLabs), and the remainder of the procedure was identical to the previously described pulldown.

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria
    Article Snippet: Paragraph title: In vitro phosphorylation. ... CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet.

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2
    Article Snippet: .. Interaction between Hjr and SIRV2gp26 coat protein To directly test the interaction between Hjr and SIRV2gp26 coat protein in vitro , anti-MBP::MBP-Hjr magnetic beads (hereafter called MBP-Hjr affinity beads) were prepared by mixing 10 µg MBP-Hjr with 0.1 mg anti-MBP magnetic beads pre-equilibrated in NEBuffer 3 (New England Biolabs). .. Bead complexes were mixed thoroughly and incubated at 4°C with shaking for 1 hour.

    Concentration Assay:

    Article Title: A conserved membrane-binding domain targets proteins to organelle contact sites
    Article Snippet: Liposomes with various lipid composition and a trace amount of [3 H]triolein (American Radiolabled Chemicals) were added to the magnetic beads to a final concentration of 1 mM in a total volume 100 μl and incubated at 30°C for 30 minutes. .. The amount of MBP fusion protein was determined by immunoblotting with anti-MBP antibody (New England Biolabs) and comparing the results with those of known amounts of pure MBP.

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201
    Article Snippet: The culture was incubated with shaking at 28°C for 15 min. To quench the cross-linking reaction, glycine was added to make a final concentration of 250 mM, followed by a 15 min incubation at room temperature. .. The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples.

    Recombinant:

    Article Title: Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1
    Article Snippet: For each nucleosome pulldown in , 5 µL streptavidin-coated beads (Pierce) were incubated with 5 pmol recombinant H3K9me2-UnDNA 187-bp biotinylated nucleosomes for 30 min at RT. .. For Western blot, 10 µL of bound protein and 2% of the unbound fraction was loaded for input onto SDS/PAGE.), 5 pmol of MBP-DNMT1 was bound to 5 µL of anti-MBP magnetic beads (New England BioLabs), and the remainder of the procedure was identical to the previously described pulldown.

    Variant Assay:

    Article Title: Understanding the role of the Q338H MUTYH variant in oxidative damage repair
    Article Snippet: Wild-type and variant MBP–MUTYH proteins (5 µg) were incubated with HeLa nuclear extracts overnight at 4°C in HEPES–KOH 10 mM, pH 7.4, KCl 100 mM and MgCl2 10 mM buffer. .. The samples were then added to 30 µl of anti-MBP magnetic beads (1 mg/ml and binding capacity of 10 µg/mg) (New England BioLabs, Ipswich, MA, USA), pre-treated as described by manufacturer.

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    New England Biolabs anti mbp magnetic beads
    <t>MrrA</t> phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the <t>MBP-CC2874</t> preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.
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    MrrA phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the MBP-CC2874 preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.

    Journal: mBio

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria

    doi: 10.1128/mBio.00809-18

    Figure Lengend Snippet: MrrA phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the MBP-CC2874 preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.

    Article Snippet: CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet.

    Techniques: SDS Page, Autoradiography, Activity Assay, Purification

    LovK and PhyK are phosphotransferases that are activated by MrrA~P. (A) Schematic representation of two possible modes of action of MrrA~P, phosphotransfer to and allosteric activation of PhyK and LovK. For reasons of simplicity, only PhyK is shown. (B) PhyK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, the kinase CC2874, MrrA, and different PhyK variants. Purified PhyK wild type or mutant variants harboring mutations in the G1 (G514A/G516A) or G2 (G526A) box of the ATP-binding site were used as indicated. The positions of phosphorylated proteins on the gel are indicated on the right. (C) LovK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, kinase CC2874, MrrA, and different LovK variants. Purified LovK wild type or mutant variants harboring mutations in the G1 (G319A/G321A) or G2 (G332A) box ATP-binding site of the CA domain were used as indicated. MrrA dilution factors are indicated in each lane. The positions of phosphorylated proteins on the gel are indicated on the right. (D) Phosphotransfer to PhyR does not require a conserved CA domain. Phosphorylation reaction mixtures containing radiolabeled ATP, kinase CC2874, MrrA, PhyR, NepR, and different PhyK variants are as in panel C. The positions of phosphorylated proteins on the gel are indicated on the right. (E) Preparation of purified MrrA~P. Kinase CC2874 alone or with MrrA was phosphorylated with radiolabeled ATP (lanes 1 and 7), and CC2874 was subsequently removed using anti-MBP magnetic beads (lanes 2 and 8). Next, ATP was hydrolyzed by treating mixtures with hexokinase and glucose (lanes 3 and 9). CC2874 was added back to ATP-depleted samples, and mixtures were incubated for 0.5, 1.0, and 5 min (lanes 4 to 6 and 10 to 12). (F) Purified MrrA~P transfers phosphate to LovK. MrrA~P was prepared as in panel E (lane 1), CC2874 was removed (lane 2), and ATP was degraded (lane 3). Fresh CC2874 (lane 4) or LovK was added, and phosphotransfer from MrrA~P was monitored after 10 s, 20 s, 1 min, 2 min, 10 min, and 20 min (lanes 5 to 10, respectively). (G) ATP binding is not required for PhyK activity in the general stress response. Δ lovK strains harboring an empty vector (EV) or a plasmid expressing different phyK alleles from a cumate-inducible promoter were analyzed. Plasmid-driven variants of PhyK contained mutations in the G1 or G2 box of the ATP-binding pocket (see above) or in the conserved phosphoacceptor His371. SigT-dependent sigU promoter activity (Miller units) was determined using a lacZ promoter fusion in strains grown in the presence (+) or absence (−) of cumate. PhyK variants harbored a C-terminal 3×FLAG tag that allowed monitoring their expression by immunoblot analysis (lower panels). An immunoblot with anti-MreB antibodies is shown as a control. Note that the sigUp-lacZ reporter fusion used in these experiments differed from the one used in experiments above ( Fig. 1D ) and shows higher basal activity (compare wild-type PhyK and empty-vector control in panel G).

    Journal: mBio

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria

    doi: 10.1128/mBio.00809-18

    Figure Lengend Snippet: LovK and PhyK are phosphotransferases that are activated by MrrA~P. (A) Schematic representation of two possible modes of action of MrrA~P, phosphotransfer to and allosteric activation of PhyK and LovK. For reasons of simplicity, only PhyK is shown. (B) PhyK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, the kinase CC2874, MrrA, and different PhyK variants. Purified PhyK wild type or mutant variants harboring mutations in the G1 (G514A/G516A) or G2 (G526A) box of the ATP-binding site were used as indicated. The positions of phosphorylated proteins on the gel are indicated on the right. (C) LovK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, kinase CC2874, MrrA, and different LovK variants. Purified LovK wild type or mutant variants harboring mutations in the G1 (G319A/G321A) or G2 (G332A) box ATP-binding site of the CA domain were used as indicated. MrrA dilution factors are indicated in each lane. The positions of phosphorylated proteins on the gel are indicated on the right. (D) Phosphotransfer to PhyR does not require a conserved CA domain. Phosphorylation reaction mixtures containing radiolabeled ATP, kinase CC2874, MrrA, PhyR, NepR, and different PhyK variants are as in panel C. The positions of phosphorylated proteins on the gel are indicated on the right. (E) Preparation of purified MrrA~P. Kinase CC2874 alone or with MrrA was phosphorylated with radiolabeled ATP (lanes 1 and 7), and CC2874 was subsequently removed using anti-MBP magnetic beads (lanes 2 and 8). Next, ATP was hydrolyzed by treating mixtures with hexokinase and glucose (lanes 3 and 9). CC2874 was added back to ATP-depleted samples, and mixtures were incubated for 0.5, 1.0, and 5 min (lanes 4 to 6 and 10 to 12). (F) Purified MrrA~P transfers phosphate to LovK. MrrA~P was prepared as in panel E (lane 1), CC2874 was removed (lane 2), and ATP was degraded (lane 3). Fresh CC2874 (lane 4) or LovK was added, and phosphotransfer from MrrA~P was monitored after 10 s, 20 s, 1 min, 2 min, 10 min, and 20 min (lanes 5 to 10, respectively). (G) ATP binding is not required for PhyK activity in the general stress response. Δ lovK strains harboring an empty vector (EV) or a plasmid expressing different phyK alleles from a cumate-inducible promoter were analyzed. Plasmid-driven variants of PhyK contained mutations in the G1 or G2 box of the ATP-binding pocket (see above) or in the conserved phosphoacceptor His371. SigT-dependent sigU promoter activity (Miller units) was determined using a lacZ promoter fusion in strains grown in the presence (+) or absence (−) of cumate. PhyK variants harbored a C-terminal 3×FLAG tag that allowed monitoring their expression by immunoblot analysis (lower panels). An immunoblot with anti-MreB antibodies is shown as a control. Note that the sigUp-lacZ reporter fusion used in these experiments differed from the one used in experiments above ( Fig. 1D ) and shows higher basal activity (compare wild-type PhyK and empty-vector control in panel G).

    Article Snippet: CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet.

    Techniques: Activation Assay, Purification, Mutagenesis, Binding Assay, Magnetic Beads, Incubation, Activity Assay, Plasmid Preparation, Expressing

    SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.

    Journal: PLoS ONE

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2

    doi: 10.1371/journal.pone.0023668

    Figure Lengend Snippet: SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.

    Article Snippet: Interaction between Hjr and SIRV2gp26 coat protein To directly test the interaction between Hjr and SIRV2gp26 coat protein in vitro , anti-MBP::MBP-Hjr magnetic beads (hereafter called MBP-Hjr affinity beads) were prepared by mixing 10 µg MBP-Hjr with 0.1 mg anti-MBP magnetic beads pre-equilibrated in NEBuffer 3 (New England Biolabs).

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Sequencing, Construct, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

    SIRV2 Hjr interacts with SIRV2gp26 coat protein in vitro . The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.

    Journal: PLoS ONE

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2

    doi: 10.1371/journal.pone.0023668

    Figure Lengend Snippet: SIRV2 Hjr interacts with SIRV2gp26 coat protein in vitro . The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.

    Article Snippet: Interaction between Hjr and SIRV2gp26 coat protein To directly test the interaction between Hjr and SIRV2gp26 coat protein in vitro , anti-MBP::MBP-Hjr magnetic beads (hereafter called MBP-Hjr affinity beads) were prepared by mixing 10 µg MBP-Hjr with 0.1 mg anti-MBP magnetic beads pre-equilibrated in NEBuffer 3 (New England Biolabs).

    Techniques: In Vitro, Immunoprecipitation, SDS Page, Staining

    ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P

    Journal: Frontiers in Microbiology

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201

    doi: 10.3389/fmicb.2018.01584

    Figure Lengend Snippet: ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P

    Article Snippet: The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, In Vivo, Polymerase Chain Reaction, Generated, Immunoprecipitation, Isolation, Magnetic Beads, Quantitative RT-PCR, Derivative Assay

    ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P

    Journal: Frontiers in Microbiology

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201

    doi: 10.3389/fmicb.2018.01584

    Figure Lengend Snippet: ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P

    Article Snippet: The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, In Vivo, Polymerase Chain Reaction, Generated, Immunoprecipitation, Isolation, Magnetic Beads, Quantitative RT-PCR, Derivative Assay