Structured Review

Promega t4 dna ligase buffer
DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with <t>T4</t> DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.
T4 Dna Ligase Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/t4 dna ligase buffer/product/Promega
Average 93 stars, based on 12 article reviews
Price from $9.99 to $1999.99
t4 dna ligase buffer - by Bioz Stars, 2020-09
93/100 stars

Images

1) Product Images from "CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions"

Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023572

DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.
Figure Legend Snippet: DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.

Techniques Used: Plasmid Preparation, Incubation, Recombinant, Staining, Autoradiography, Labeling, Ligation, DNA Ligation, Electrophoresis

2) Product Images from "The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus"

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040192

DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.
Figure Legend Snippet: DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

Techniques Used: Recombinant, Binding Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining, Labeling, Ligation, DNA Ligation, Electrophoresis, Autoradiography

DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.
Figure Legend Snippet: DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

Techniques Used: Incubation, Ligation, DNA Ligation, Electrophoresis, Autoradiography

Related Articles

Amplification:

Article Title: Programmed Evolution for Optimization of Orthogonal Metabolic Output in Bacteria
Article Snippet: .. J-GGA was carried out in 10 µL reactions containing 50 ng amplified plasmid, an equimolar quantity of the desired insert, 1 µL 10 T4 DNA Ligase buffer (Promega), 0.5 µL (10 units) BsaI (New England Biolabs), and 0.5 µL (1 Weiss unit) T4 DNA Ligase (Promega). .. Products were transformed into JM109 E . coli (Zymo Research) and plated on LB agar, Lennox (Accumedia, Neogen Corp.) with 50 µg/mL ampicillin.

Concentration Assay:

Article Title: Role of auxin during intercellular infection of Discaria trinervis by Frankia
Article Snippet: .. The reaction mix was completed with 15 units of high concentration HC T4 DNA ligase (Promega), 2.5 units of BsaI (NEB), 10 × T4 DNA ligase buffer (Promega) and water up to 15 μ L. The restriction-ligation reaction was performed in a thermocycler with the following program: 50 cycles of 2 min incubation at 37°C and 5 min at 16°C, followed by 5 min incubation at 50°C and then a heat inactivation at 80°C for another 5 min. 5 μ l of the reaction were used to transformed E. coli TOP10 chemically competent cells by heat shock. .. Composite plants generation The pDISC-ProDtAUX1:GUS and the DR5:Venus binary vectors were introduced into A. rhizogenes ARqua1 strain by electroporation.

Incubation:

Article Title: Role of auxin during intercellular infection of Discaria trinervis by Frankia
Article Snippet: .. The reaction mix was completed with 15 units of high concentration HC T4 DNA ligase (Promega), 2.5 units of BsaI (NEB), 10 × T4 DNA ligase buffer (Promega) and water up to 15 μ L. The restriction-ligation reaction was performed in a thermocycler with the following program: 50 cycles of 2 min incubation at 37°C and 5 min at 16°C, followed by 5 min incubation at 50°C and then a heat inactivation at 80°C for another 5 min. 5 μ l of the reaction were used to transformed E. coli TOP10 chemically competent cells by heat shock. .. Composite plants generation The pDISC-ProDtAUX1:GUS and the DR5:Venus binary vectors were introduced into A. rhizogenes ARqua1 strain by electroporation.

DNA Purification:

Article Title: Modulating the bioluminescence emission of photoproteins by in vivo site-directed incorporation of non-natural amino acids
Article Snippet: .. T4 DNA ligase and T4 DNA ligase buffer was purchased from Promega, exTaq DNA polymerase from Takara, plasmid DNA purification and gel extraction kits from Qiagen, and tris(hydroxymethyl)amino methane (Tris) free base, ethylenediaminetetraacetic acid (EDTA) sodium salt, glucose, sodium dodecyl sulfate (SDS), ampicillin, tetracycline, and arabinose were purchased from Sigma. .. All other components necessary for arabinose auto-induction media were obtained from Sigma or DIFCO.

Polymerase Chain Reaction:

Article Title: Genome-wide identification of microsatellites in white clover (Trifolium repens L.) using FIASCO and phpSSRMiner
Article Snippet: .. An initial PCR reaction in a 25 μl reaction contained 250 ng of genomic DNA, 25 μM of NaCl, 1 unit of T4 ligase (Promega, Madison, WI, USA), 1× T4 DNA ligase buffer (Promega, Madison, WI, USA), 25 μg/ml BSA (New England Biolabs, Boston, MA, USA), 2.5 units of Mse I, and 2 pmol of adapter. .. To make the adapters, 1 μM of each of two oligonucleotides (5' GACGATGAGTCCTGAG 3' and 5' TACTCAGGACTCAT 3') were combined and heated at 95°C for 5 min in a MJ Research thermal cycler.

Gel Extraction:

Article Title: Modulating the bioluminescence emission of photoproteins by in vivo site-directed incorporation of non-natural amino acids
Article Snippet: .. T4 DNA ligase and T4 DNA ligase buffer was purchased from Promega, exTaq DNA polymerase from Takara, plasmid DNA purification and gel extraction kits from Qiagen, and tris(hydroxymethyl)amino methane (Tris) free base, ethylenediaminetetraacetic acid (EDTA) sodium salt, glucose, sodium dodecyl sulfate (SDS), ampicillin, tetracycline, and arabinose were purchased from Sigma. .. All other components necessary for arabinose auto-induction media were obtained from Sigma or DIFCO.

Transformation Assay:

Article Title: Role of auxin during intercellular infection of Discaria trinervis by Frankia
Article Snippet: .. The reaction mix was completed with 15 units of high concentration HC T4 DNA ligase (Promega), 2.5 units of BsaI (NEB), 10 × T4 DNA ligase buffer (Promega) and water up to 15 μ L. The restriction-ligation reaction was performed in a thermocycler with the following program: 50 cycles of 2 min incubation at 37°C and 5 min at 16°C, followed by 5 min incubation at 50°C and then a heat inactivation at 80°C for another 5 min. 5 μ l of the reaction were used to transformed E. coli TOP10 chemically competent cells by heat shock. .. Composite plants generation The pDISC-ProDtAUX1:GUS and the DR5:Venus binary vectors were introduced into A. rhizogenes ARqua1 strain by electroporation.

Recombinant:

Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions
Article Snippet: .. Briefly, a 32 P-labeled-66-bp or a 32 P-labeled-123-bp DNA fragments (1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (50 ng), total (10 µg), nuclear (4 µg) or cytoplasmic (4 µg) adult worm extracts, in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µl. .. The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min.

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus
Article Snippet: .. Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL. .. The DNA was then ligated with T4 DNA ligase (0.6 unit/reaction; Promega) at 30°C for 30 min, and the ligation reactions were terminated by incubation of samples at 65°C for 15 min.

Plasmid Preparation:

Article Title: Programmed Evolution for Optimization of Orthogonal Metabolic Output in Bacteria
Article Snippet: .. J-GGA was carried out in 10 µL reactions containing 50 ng amplified plasmid, an equimolar quantity of the desired insert, 1 µL 10 T4 DNA Ligase buffer (Promega), 0.5 µL (10 units) BsaI (New England Biolabs), and 0.5 µL (1 Weiss unit) T4 DNA Ligase (Promega). .. Products were transformed into JM109 E . coli (Zymo Research) and plated on LB agar, Lennox (Accumedia, Neogen Corp.) with 50 µg/mL ampicillin.

Article Title: Engineering a Model Cell for Rational Tuning of GPCR Signaling
Article Snippet: .. Golden Gate reactions were prepared as follows: 0.1 μL of backbone vector, 0.5 μL of each plasmid, 1 μL T4 DNA ligase buffer (Promega), 0.5 μL T7 DNA Ligase (NEB), 0.5 μL restriction enzyme (BsaI or BsmBI) (NEB), and water to bring the final volume to 10 μL. .. Reaction mixtures were then incubated in a thermocycler using the following program: (42°C for 2 min, 16°C for 5 min) x 25 cycles, followed by a final digestion step of 60°C for 10 min, and then heat inactivation at 80°C for 10 min.

Article Title: Modulating the bioluminescence emission of photoproteins by in vivo site-directed incorporation of non-natural amino acids
Article Snippet: .. T4 DNA ligase and T4 DNA ligase buffer was purchased from Promega, exTaq DNA polymerase from Takara, plasmid DNA purification and gel extraction kits from Qiagen, and tris(hydroxymethyl)amino methane (Tris) free base, ethylenediaminetetraacetic acid (EDTA) sodium salt, glucose, sodium dodecyl sulfate (SDS), ampicillin, tetracycline, and arabinose were purchased from Sigma. .. All other components necessary for arabinose auto-induction media were obtained from Sigma or DIFCO.

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    Promega t4 dna ligase buffer
    DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with <t>T4</t> DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.
    T4 Dna Ligase Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase buffer/product/Promega
    Average 93 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase buffer - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    92
    Promega t4 ligase buffer
    DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with <t>T4</t> DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.
    T4 Ligase Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase buffer/product/Promega
    Average 92 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    t4 ligase buffer - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

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    DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.

    Journal: PLoS ONE

    Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

    doi: 10.1371/journal.pone.0023572

    Figure Lengend Snippet: DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.

    Article Snippet: Briefly, a 32 P-labeled-66-bp or a 32 P-labeled-123-bp DNA fragments (1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (50 ng), total (10 µg), nuclear (4 µg) or cytoplasmic (4 µg) adult worm extracts, in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µl.

    Techniques: Plasmid Preparation, Incubation, Recombinant, Staining, Autoradiography, Labeling, Ligation, DNA Ligation, Electrophoresis

    DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

    Journal: PLoS ONE

    Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

    doi: 10.1371/journal.pone.0040192

    Figure Lengend Snippet: DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

    Article Snippet: Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL.

    Techniques: Recombinant, Binding Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining, Labeling, Ligation, DNA Ligation, Electrophoresis, Autoradiography

    DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

    Journal: PLoS ONE

    Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

    doi: 10.1371/journal.pone.0040192

    Figure Lengend Snippet: DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

    Article Snippet: Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL.

    Techniques: Incubation, Ligation, DNA Ligation, Electrophoresis, Autoradiography