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    Structured Review

    New England Biolabs lic
    Experimental results for exonuclease combinations treatment. ( A ) The cleavage mechanism of Lamada and Exonuclease I combinations. Other exonuclease combinations <t>(Lambda</t> and RecJF; Exonuclease I and Exonuclease III) are shown in and . ( B ) Three exonuclease combinations (LRL; LRC; <t>LIC)</t> removed linear DNA from a paradigm mixture. M, 1 kb DNA ladder; P, pGL4.23 plasmid; L, linearized plasmid; X, mixture (plasmid and linear DNA 1:1); 1-X, LRL-Mixture, LRL to cut mixture; 2-X, LRC-Mixture, LRC to cut mixture; 3-X, LIC-Mixture, LIC to cut mixture; 3-P, LIC to cut plasmid; 3-L, LIC-Lin, LIC to cut linearized plasmid; MS, supercoiled ladder. ( C ) I+III combination and Exonuclease VIII, truncated elimination tests. 5-X, VIII4-Mixture, Exonuclease VIII, truncated within Buffer 4 to remove mixture; 6-X, VIIIC-Mixture, Exonuclease VIII, truncated within CutSmart buffer to remove mixture; 4-X: I+III-Mixture, I+III to remove mixture. Loading samples for agarose gel electrophoresis were purified by phenol-chloroform.
    Lic, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4877 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Exonuclease combinations reduce noises in 3D genomics technologies"

    Article Title: Exonuclease combinations reduce noises in 3D genomics technologies

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa106

    Experimental results for exonuclease combinations treatment. ( A ) The cleavage mechanism of Lamada and Exonuclease I combinations. Other exonuclease combinations (Lambda and RecJF; Exonuclease I and Exonuclease III) are shown in and . ( B ) Three exonuclease combinations (LRL; LRC; LIC) removed linear DNA from a paradigm mixture. M, 1 kb DNA ladder; P, pGL4.23 plasmid; L, linearized plasmid; X, mixture (plasmid and linear DNA 1:1); 1-X, LRL-Mixture, LRL to cut mixture; 2-X, LRC-Mixture, LRC to cut mixture; 3-X, LIC-Mixture, LIC to cut mixture; 3-P, LIC to cut plasmid; 3-L, LIC-Lin, LIC to cut linearized plasmid; MS, supercoiled ladder. ( C ) I+III combination and Exonuclease VIII, truncated elimination tests. 5-X, VIII4-Mixture, Exonuclease VIII, truncated within Buffer 4 to remove mixture; 6-X, VIIIC-Mixture, Exonuclease VIII, truncated within CutSmart buffer to remove mixture; 4-X: I+III-Mixture, I+III to remove mixture. Loading samples for agarose gel electrophoresis were purified by phenol-chloroform.
    Figure Legend Snippet: Experimental results for exonuclease combinations treatment. ( A ) The cleavage mechanism of Lamada and Exonuclease I combinations. Other exonuclease combinations (Lambda and RecJF; Exonuclease I and Exonuclease III) are shown in and . ( B ) Three exonuclease combinations (LRL; LRC; LIC) removed linear DNA from a paradigm mixture. M, 1 kb DNA ladder; P, pGL4.23 plasmid; L, linearized plasmid; X, mixture (plasmid and linear DNA 1:1); 1-X, LRL-Mixture, LRL to cut mixture; 2-X, LRC-Mixture, LRC to cut mixture; 3-X, LIC-Mixture, LIC to cut mixture; 3-P, LIC to cut plasmid; 3-L, LIC-Lin, LIC to cut linearized plasmid; MS, supercoiled ladder. ( C ) I+III combination and Exonuclease VIII, truncated elimination tests. 5-X, VIII4-Mixture, Exonuclease VIII, truncated within Buffer 4 to remove mixture; 6-X, VIIIC-Mixture, Exonuclease VIII, truncated within CutSmart buffer to remove mixture; 4-X: I+III-Mixture, I+III to remove mixture. Loading samples for agarose gel electrophoresis were purified by phenol-chloroform.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Purification

    Quantification effects for different combination treatments. ( A ) Results of every linear elimination treatment, measured by Qubit. Plasmid (250 ng) and linear DNA (250 ng) were mixed, as templates (X, Mixture). Linear DNA (500 ng) (3-L, LIC-Lin) was set as control treatments. Every treatment had three replicates. 1-X, LRL-Mixture; 2-X, LRC-Mixture; 3-X, LIC-Mixture; 4-X, I+III-Mixture; 5-X, VIII4-Mixture; 6-X, VIIIC-Mixture. ( B ) The qPCR results from three exonuclease combination treatments (in accordance with Figure ). The primer BH2 was used across the plasmid BamHI site. Same quality (SQ): The Qubit amounts of qPCR input for all treatments were consistent (Supplemental S.1.2). Same volume (SV): The qPCR input volumes were consistent (Supplemental S.1.4 and ). P, pGL4.23 plasmid; L, linearized plasmid; X, Mixture; 3-P, LIC to cut plasmid; 3-L, LIC to cut linearized plasmid. Three experiments for each treatment were combined for enlarging the volume before purification. ( C ) The qPCR results correspond with Figure . The primer was BH2. Data are presented as mean ± SEM.
    Figure Legend Snippet: Quantification effects for different combination treatments. ( A ) Results of every linear elimination treatment, measured by Qubit. Plasmid (250 ng) and linear DNA (250 ng) were mixed, as templates (X, Mixture). Linear DNA (500 ng) (3-L, LIC-Lin) was set as control treatments. Every treatment had three replicates. 1-X, LRL-Mixture; 2-X, LRC-Mixture; 3-X, LIC-Mixture; 4-X, I+III-Mixture; 5-X, VIII4-Mixture; 6-X, VIIIC-Mixture. ( B ) The qPCR results from three exonuclease combination treatments (in accordance with Figure ). The primer BH2 was used across the plasmid BamHI site. Same quality (SQ): The Qubit amounts of qPCR input for all treatments were consistent (Supplemental S.1.2). Same volume (SV): The qPCR input volumes were consistent (Supplemental S.1.4 and ). P, pGL4.23 plasmid; L, linearized plasmid; X, Mixture; 3-P, LIC to cut plasmid; 3-L, LIC to cut linearized plasmid. Three experiments for each treatment were combined for enlarging the volume before purification. ( C ) The qPCR results correspond with Figure . The primer was BH2. Data are presented as mean ± SEM.

    Techniques Used: Plasmid Preparation, Purification

    C-technology application and preliminary assessment in Hi-C. ( A ) DNA library (572 ng) linear noise elimination for C-technologies, after proximal-ligation. M (left), 1 kb DNA ladder; 1: LRL, LRL used for linear noise elimination. 2: LRC, LRC used for linear noise elimination; 3: LIC, LIC used for linear noise elimination; 4: I+IIIC, I+IIIC used for linear noise elimination; C: Co, without exonucleases digestion; M (right): 100 bp DNA ladder. ( B ) The yield ratio of remaining DNA after four linear elimination treatments, which was consistent to (A). The remaining ratio (gray) and eliminated ratio (purple) are shown together in the bar chart. ( C ) A 40 ng library was given four exonuclease treatment combinations, along with addition of plasmid chaperon carrier, as indicated. M: 1 kb + 100 bp DNA ladder. 1′: LRLP1, LRL with P1 chaperon used for linear noise elimination; 2′: LRC with P1 chaperon used for linear noise elimination; 3′: LICP1, LIC with P1 chaperon used for linear noise elimination; 4′: I+IIICP1, I+IIIC with P1 chaperon used for linear noise elimination; C’: P1 chaperon added, without exonucleases digestion. ( D ) The yield ratio of remaining DNA, after four linear DNA elimination treatments, which was consistent to ( C ). ( E ) HiC-Pro filtering results of Hi-C with exonuclease elimination LIC, compared with standard Hi-C in Figure . LIC-Hi-C had three experimental replicates (LIC-Hi-C 1; LIC-Hi-C 2; and LIC-Hi-C 3). HiC-Pro terms are as indicated in Supplemental S.3.
    Figure Legend Snippet: C-technology application and preliminary assessment in Hi-C. ( A ) DNA library (572 ng) linear noise elimination for C-technologies, after proximal-ligation. M (left), 1 kb DNA ladder; 1: LRL, LRL used for linear noise elimination. 2: LRC, LRC used for linear noise elimination; 3: LIC, LIC used for linear noise elimination; 4: I+IIIC, I+IIIC used for linear noise elimination; C: Co, without exonucleases digestion; M (right): 100 bp DNA ladder. ( B ) The yield ratio of remaining DNA after four linear elimination treatments, which was consistent to (A). The remaining ratio (gray) and eliminated ratio (purple) are shown together in the bar chart. ( C ) A 40 ng library was given four exonuclease treatment combinations, along with addition of plasmid chaperon carrier, as indicated. M: 1 kb + 100 bp DNA ladder. 1′: LRLP1, LRL with P1 chaperon used for linear noise elimination; 2′: LRC with P1 chaperon used for linear noise elimination; 3′: LICP1, LIC with P1 chaperon used for linear noise elimination; 4′: I+IIICP1, I+IIIC with P1 chaperon used for linear noise elimination; C’: P1 chaperon added, without exonucleases digestion. ( D ) The yield ratio of remaining DNA, after four linear DNA elimination treatments, which was consistent to ( C ). ( E ) HiC-Pro filtering results of Hi-C with exonuclease elimination LIC, compared with standard Hi-C in Figure . LIC-Hi-C had three experimental replicates (LIC-Hi-C 1; LIC-Hi-C 2; and LIC-Hi-C 3). HiC-Pro terms are as indicated in Supplemental S.3.

    Techniques Used: Hi-C, Ligation, Plasmid Preparation

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    (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with λ-phosphatase for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .

    Journal: PLOS One

    Article Title: Mechanistic studies of PFKFB2 reveal a novel inhibitor of its kinase activity

    doi: 10.1371/journal.pone.0317167

    Figure Lengend Snippet: (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with λ-phosphatase for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .

    Article Snippet: Assay conditions were 100 µL sonicated sample, 10 µL MnCl 2 (10 mM NEB), 10 µL phosphatase buffer (10x PMP buffer, NEB) with or without 2 µL Lambda protein phosphatase (NEB, 400000 U/mL).

    Techniques: Western Blot, Staining, Expressing, Purification, In Vitro, Phospho-proteomics, Incubation, Mutagenesis, Extraction, Bacteria, Lysis, Quantitation Assay