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    Structured Review

    New England Biolabs bsa
    A direct physical interaction between <t>RECQ1</t> and Ku70/80 in vitro . A. RECQ1 directly interacts with Ku70 and Ku80. GST or GST fused-full length RECQ1 was incubated with bacterially expressed Ku70/80, Ku70, Ku80 or N-Ku80 (lacking the C-terminus amino acid residues 565–732) followed by extensive washing of the beads, SDS-PAGE, and Western transfer. The blots were probed separately with anti-His (for Ku detection) and anti-RECQ1 antibodies. Input lanes account for 10% of the bacterial lysate expressing Ku protein used in the pull-down reactions. B. Recombinant RECQ1 and Ku70/80 proteins interact directly as shown by ELISA. Either <t>BSA</t> or purified recombinant Ku70/80 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant RECQ1 (0–50 nM) for 1 h at 30°C. Parallel wells contained DNaseI (100 U/ml) or EtBr (50 µg/ml) in the binding step to test for DNA-mediated protein interaction. Following washing, Ku70/80-bound RECQ1 was detected by ELISA using anti-RECQ1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. C. GST alone or GST-RECQ1 fragments (as indicated) bound to glutathione beads were incubated overnight at 4°C with HeLa extract (500 µg) that was either untreated or pre-treated with benzonase. After extensive washings, the bound Ku70/80 was eluted with SDS sample buffer and analyzed by Western blot using anti-Ku70 and anti-Ku80 antibodies (right). Coomassie staining of the eluted proteins was done to test expression of various GST-fusion fragments of RECQ1 (left). GST-RECQ1 proteins are marked by asterisk. Marker, protein molecular weight marker.
    Bsa, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsa - by Bioz Stars, 2020-09
    92/100 stars

    Images

    1) Product Images from "Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks"

    Article Title: Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062481

    A direct physical interaction between RECQ1 and Ku70/80 in vitro . A. RECQ1 directly interacts with Ku70 and Ku80. GST or GST fused-full length RECQ1 was incubated with bacterially expressed Ku70/80, Ku70, Ku80 or N-Ku80 (lacking the C-terminus amino acid residues 565–732) followed by extensive washing of the beads, SDS-PAGE, and Western transfer. The blots were probed separately with anti-His (for Ku detection) and anti-RECQ1 antibodies. Input lanes account for 10% of the bacterial lysate expressing Ku protein used in the pull-down reactions. B. Recombinant RECQ1 and Ku70/80 proteins interact directly as shown by ELISA. Either BSA or purified recombinant Ku70/80 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant RECQ1 (0–50 nM) for 1 h at 30°C. Parallel wells contained DNaseI (100 U/ml) or EtBr (50 µg/ml) in the binding step to test for DNA-mediated protein interaction. Following washing, Ku70/80-bound RECQ1 was detected by ELISA using anti-RECQ1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. C. GST alone or GST-RECQ1 fragments (as indicated) bound to glutathione beads were incubated overnight at 4°C with HeLa extract (500 µg) that was either untreated or pre-treated with benzonase. After extensive washings, the bound Ku70/80 was eluted with SDS sample buffer and analyzed by Western blot using anti-Ku70 and anti-Ku80 antibodies (right). Coomassie staining of the eluted proteins was done to test expression of various GST-fusion fragments of RECQ1 (left). GST-RECQ1 proteins are marked by asterisk. Marker, protein molecular weight marker.
    Figure Legend Snippet: A direct physical interaction between RECQ1 and Ku70/80 in vitro . A. RECQ1 directly interacts with Ku70 and Ku80. GST or GST fused-full length RECQ1 was incubated with bacterially expressed Ku70/80, Ku70, Ku80 or N-Ku80 (lacking the C-terminus amino acid residues 565–732) followed by extensive washing of the beads, SDS-PAGE, and Western transfer. The blots were probed separately with anti-His (for Ku detection) and anti-RECQ1 antibodies. Input lanes account for 10% of the bacterial lysate expressing Ku protein used in the pull-down reactions. B. Recombinant RECQ1 and Ku70/80 proteins interact directly as shown by ELISA. Either BSA or purified recombinant Ku70/80 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant RECQ1 (0–50 nM) for 1 h at 30°C. Parallel wells contained DNaseI (100 U/ml) or EtBr (50 µg/ml) in the binding step to test for DNA-mediated protein interaction. Following washing, Ku70/80-bound RECQ1 was detected by ELISA using anti-RECQ1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. C. GST alone or GST-RECQ1 fragments (as indicated) bound to glutathione beads were incubated overnight at 4°C with HeLa extract (500 µg) that was either untreated or pre-treated with benzonase. After extensive washings, the bound Ku70/80 was eluted with SDS sample buffer and analyzed by Western blot using anti-Ku70 and anti-Ku80 antibodies (right). Coomassie staining of the eluted proteins was done to test expression of various GST-fusion fragments of RECQ1 (left). GST-RECQ1 proteins are marked by asterisk. Marker, protein molecular weight marker.

    Techniques Used: In Vitro, Incubation, SDS Page, Western Blot, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay, Binding Assay, Staining, Marker, Molecular Weight

    2) Product Images from "The Ska complex promotes Aurora B activity to ensure chromosome biorientation"

    Article Title: The Ska complex promotes Aurora B activity to ensure chromosome biorientation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201603019

    The Ska complex promotes the catalytic activity of Aurora B in vitro. (A) Time course kinase assay with Aurora B–His and MBP–INCENP 790–919 –His preincubated with Ska complex or equimolar amounts of BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (B) Quantification of histone H3 32 P signals from A. Signals were normalized to H3 and Aurora B protein levels monitored by Ponceau S staining (Ponc. S) and Western blotting (WB), respectively. Signal intensities are expressed relative to the first time-point. Data represent mean ± SD (three experiments). (C) Aurora B–His was incubated with recombinant Ska complex before pull-down with beads coupled to anti-Ska1 antibody or control antibody (IgG). UB, unbound fraction; B, bound fraction. (D) Immunoprecipitates (IP) from mitotic HeLa S3 cell extracts, obtained using anti-Ska1 antibodies or control antibodies (IgG), analyzed by WB. (E) Aurora B–His was preincubated with Ska complex (comp.) or histone H3, as control, before incubation with γ-[ 32 P]ATP. Aurora B autophosphorylation is visualized by autoradiography ( 32 P) and Aurora B levels by Coomassie Brilliant Blue (CBB) staining (see Fig. S5 G for uncropped results). (F) Quantification of Aurora B autophosphorylation signals from E (one experiment). Signal intensities are expressed relative to the first concentration. (G) Time course kinase assay with recombinant Aurora B–His preincubated with Ska complex, equimolar amounts of MBP–INCENP 790–919 –His or BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (H) Quantification of Aurora B kinase activity as in B (one experiment).
    Figure Legend Snippet: The Ska complex promotes the catalytic activity of Aurora B in vitro. (A) Time course kinase assay with Aurora B–His and MBP–INCENP 790–919 –His preincubated with Ska complex or equimolar amounts of BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (B) Quantification of histone H3 32 P signals from A. Signals were normalized to H3 and Aurora B protein levels monitored by Ponceau S staining (Ponc. S) and Western blotting (WB), respectively. Signal intensities are expressed relative to the first time-point. Data represent mean ± SD (three experiments). (C) Aurora B–His was incubated with recombinant Ska complex before pull-down with beads coupled to anti-Ska1 antibody or control antibody (IgG). UB, unbound fraction; B, bound fraction. (D) Immunoprecipitates (IP) from mitotic HeLa S3 cell extracts, obtained using anti-Ska1 antibodies or control antibodies (IgG), analyzed by WB. (E) Aurora B–His was preincubated with Ska complex (comp.) or histone H3, as control, before incubation with γ-[ 32 P]ATP. Aurora B autophosphorylation is visualized by autoradiography ( 32 P) and Aurora B levels by Coomassie Brilliant Blue (CBB) staining (see Fig. S5 G for uncropped results). (F) Quantification of Aurora B autophosphorylation signals from E (one experiment). Signal intensities are expressed relative to the first concentration. (G) Time course kinase assay with recombinant Aurora B–His preincubated with Ska complex, equimolar amounts of MBP–INCENP 790–919 –His or BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (H) Quantification of Aurora B kinase activity as in B (one experiment).

    Techniques Used: Activity Assay, In Vitro, Kinase Assay, Staining, Western Blot, Incubation, Recombinant, Autoradiography, Concentration Assay

    3) Product Images from "Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System"

    Article Title: Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00699

    Diagram of the modified Gln-tRNA-gRNA cassettes in pUC19-tRp-gRNA and pCas9-tRp-gRNA vectors. The blue arrow on the left is the Gln-tRNA promoter that is followed by the gRNA spacer insert site, gRNA scaffold (green), and RNA polyIII terminator (purple) sequences. The gRNA spacers are synthesized by annealing the sense and antisense oligonucleotides with 5′-ACCT and 3′-CAAA overhangs and inserted into the gRNA spacer insertion site of Bsa I-digested pUC19-tRp-gRNA (upper) or Bsm BI-digested pCas9-tRp-gRNA (lower). The gRNA start site is marked with a red arrow. The cleavage sites of type II restriction enzyme Bsa I and Bsm BI are marked with black arrows.
    Figure Legend Snippet: Diagram of the modified Gln-tRNA-gRNA cassettes in pUC19-tRp-gRNA and pCas9-tRp-gRNA vectors. The blue arrow on the left is the Gln-tRNA promoter that is followed by the gRNA spacer insert site, gRNA scaffold (green), and RNA polyIII terminator (purple) sequences. The gRNA spacers are synthesized by annealing the sense and antisense oligonucleotides with 5′-ACCT and 3′-CAAA overhangs and inserted into the gRNA spacer insertion site of Bsa I-digested pUC19-tRp-gRNA (upper) or Bsm BI-digested pCas9-tRp-gRNA (lower). The gRNA start site is marked with a red arrow. The cleavage sites of type II restriction enzyme Bsa I and Bsm BI are marked with black arrows.

    Techniques Used: Modification, Synthesized

    4) Product Images from "RNA Chaperone Function of a Universal Stress Protein in Arabidopsis Confers Enhanced Cold Stress Tolerance in Plants"

    Article Title: RNA Chaperone Function of a Universal Stress Protein in Arabidopsis Confers Enhanced Cold Stress Tolerance in Plants

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18122546

    Nucleic acid-binding activity of AtUSP in vitro. Indicated amounts of purified recombinant AtUSP protein were incubated with either ( A ) M13mp8 ssDNA, ( B ) M13mp8 dsDNA, or ( C ) in vitro transcribed luciferase ( luc ) mRNA. To analyze the effect of AtUSP in RNA mobility and the AtUSP-RNA complexes, 0.8% agarose gels were used for gel-shift assays. Bovine serum albumin (BSA) protein (100 μg/μL) was used as a negative control. SM presents size marker from Thermo Scientific Company.
    Figure Legend Snippet: Nucleic acid-binding activity of AtUSP in vitro. Indicated amounts of purified recombinant AtUSP protein were incubated with either ( A ) M13mp8 ssDNA, ( B ) M13mp8 dsDNA, or ( C ) in vitro transcribed luciferase ( luc ) mRNA. To analyze the effect of AtUSP in RNA mobility and the AtUSP-RNA complexes, 0.8% agarose gels were used for gel-shift assays. Bovine serum albumin (BSA) protein (100 μg/μL) was used as a negative control. SM presents size marker from Thermo Scientific Company.

    Techniques Used: Binding Assay, Activity Assay, In Vitro, Purification, Recombinant, Incubation, Luciferase, Electrophoretic Mobility Shift Assay, Negative Control, Marker

    5) Product Images from "Electrochemical Assay for the Signal-on Detection of Human DNA Methyltransferase Activity"

    Article Title: Electrochemical Assay for the Signal-on Detection of Human DNA Methyltransferase Activity

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja4085918

    Protease treatment restores peak sharpness for samples of high protein content. Chips were modified in all quadrants with the hemimethylated BssH II 22-mer. One half of the chip was treated with 100 nM Dnmt1 with 100 μg/mL BSA and 160 μM
    Figure Legend Snippet: Protease treatment restores peak sharpness for samples of high protein content. Chips were modified in all quadrants with the hemimethylated BssH II 22-mer. One half of the chip was treated with 100 nM Dnmt1 with 100 μg/mL BSA and 160 μM

    Techniques Used: Modification, Chromatin Immunoprecipitation

    6) Product Images from "Structural and Functional Characterization of IS679 and IS66-Family Elements"

    Article Title: Structural and Functional Characterization of IS679 and IS66-Family Elements

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.183.14.4296-4304.2001

    (A) Schematic representation of the IS 679 structure. IS 679 (2,704 bp) has imperfect 25-bp IRs. The IRs at the left and right inverted repeats (IRL and IRR) are indicated by solid triangles. Open, dotted, and cross-hatched arrows indicate, respectively, tnpA, tnpB , and tnpC . The two cross-hatched ovals flanking IS 679 indicate direct repeats of an 8-bp target site sequence. (B) Schematic representations of the structures of pHAN plasmids. pHAN103 carries Tn 679 with the kanamycin resistance gene (Km r ) between an intact IS 679 sequence and the 3′-end region having IRR. Plasmids pHAN104, pHAN105, and pHAN106 carry a Tn 679 derivative with deletions (hatched box) in tnpA, tnpB , and tnpC (thin arrows), respectively. Small solid arrows beneath the pHAN plasmid indicate primers used to construct each plasmid (see Materials and Methods). Primers with a tail indicate an additional sequence with a restriction site. s, Sac II; ai, Bsa I; ei, Bsp EI; gi, Bsr GI; r, Rsr II.
    Figure Legend Snippet: (A) Schematic representation of the IS 679 structure. IS 679 (2,704 bp) has imperfect 25-bp IRs. The IRs at the left and right inverted repeats (IRL and IRR) are indicated by solid triangles. Open, dotted, and cross-hatched arrows indicate, respectively, tnpA, tnpB , and tnpC . The two cross-hatched ovals flanking IS 679 indicate direct repeats of an 8-bp target site sequence. (B) Schematic representations of the structures of pHAN plasmids. pHAN103 carries Tn 679 with the kanamycin resistance gene (Km r ) between an intact IS 679 sequence and the 3′-end region having IRR. Plasmids pHAN104, pHAN105, and pHAN106 carry a Tn 679 derivative with deletions (hatched box) in tnpA, tnpB , and tnpC (thin arrows), respectively. Small solid arrows beneath the pHAN plasmid indicate primers used to construct each plasmid (see Materials and Methods). Primers with a tail indicate an additional sequence with a restriction site. s, Sac II; ai, Bsa I; ei, Bsp EI; gi, Bsr GI; r, Rsr II.

    Techniques Used: Sequencing, Plasmid Preparation, Construct

    7) Product Images from "Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System"

    Article Title: Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00699

    Diagram of the modified Gln-tRNA-gRNA cassettes in pUC19-tRp-gRNA and pCas9-tRp-gRNA vectors. The blue arrow on the left is the Gln-tRNA promoter that is followed by the gRNA spacer insert site, gRNA scaffold (green), and RNA polyIII terminator (purple) sequences. The gRNA spacers are synthesized by annealing the sense and antisense oligonucleotides with 5′-ACCT and 3′-CAAA overhangs and inserted into the gRNA spacer insertion site of Bsa I-digested pUC19-tRp-gRNA (upper) or Bsm BI-digested pCas9-tRp-gRNA (lower). The gRNA start site is marked with a red arrow. The cleavage sites of type II restriction enzyme Bsa I and Bsm BI are marked with black arrows.
    Figure Legend Snippet: Diagram of the modified Gln-tRNA-gRNA cassettes in pUC19-tRp-gRNA and pCas9-tRp-gRNA vectors. The blue arrow on the left is the Gln-tRNA promoter that is followed by the gRNA spacer insert site, gRNA scaffold (green), and RNA polyIII terminator (purple) sequences. The gRNA spacers are synthesized by annealing the sense and antisense oligonucleotides with 5′-ACCT and 3′-CAAA overhangs and inserted into the gRNA spacer insertion site of Bsa I-digested pUC19-tRp-gRNA (upper) or Bsm BI-digested pCas9-tRp-gRNA (lower). The gRNA start site is marked with a red arrow. The cleavage sites of type II restriction enzyme Bsa I and Bsm BI are marked with black arrows.

    Techniques Used: Modification, Synthesized

    8) Product Images from "Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA"

    Article Title: Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2010.07292.x

    CbpA protects plasmid DNA from degradation by nucleases. A. The panel shows naked plasmid and complexes with CbpA run on a 1% agarose gel. Plasmid (30 ng) was pre-incubated with 0, 0.5, 1.0, or 2.0 µM CbpA. Note that only every other lane has been loaded on the gel. B. The panel shows plasmid run on a 1% agarose gel. Plasmids (77 ng) were treated with different combinations of DNase I, BSA (1, 2 or 3 µM) and CbpA (1, 2 or 3 µM).
    Figure Legend Snippet: CbpA protects plasmid DNA from degradation by nucleases. A. The panel shows naked plasmid and complexes with CbpA run on a 1% agarose gel. Plasmid (30 ng) was pre-incubated with 0, 0.5, 1.0, or 2.0 µM CbpA. Note that only every other lane has been loaded on the gel. B. The panel shows plasmid run on a 1% agarose gel. Plasmids (77 ng) were treated with different combinations of DNase I, BSA (1, 2 or 3 µM) and CbpA (1, 2 or 3 µM).

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

    Related Articles

    Clone Assay:

    Article Title: Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System
    Article Snippet: .. A short cassette with two Bsm BI sites was generated by annealing the sense (LINK-SEQ-F) and antisense (LINK-SEQ-R) oligonucleotides (Supplementary Table ) and inserted between the two Bsa I sites of pUC19-tRp-gRNA by Golden Gate cloning (New England Biolabs, Ipswich, MA, United States) as described ( ; ) to generate the pUC19- LINK vector. .. The PGln-tRNA -gRNA cassette flanked by two Bsm BI sites was then released from pUC19-LINK and cloned between the Kpn I and Eco RI sites of pCRISPR/Cas-U6-1 ( ) to generate the pCas9-tRp-gRNA vector.

    Article Title: Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System
    Article Snippet: .. The resulting gRNA spacers were cloned between the two Bsa I sites of pUC19-tRp-gRNA or the two Bsm BI sites of pCas9-tRp-gRNA by Golden Gate cloning (New England Biolabs). .. Generation of the USTA Gene Replacement Constructs and Mutants The 1.01-kb upstream and 1.09-kb downstream flanking sequences of USTA were amplified with primer pairs of USTA1F/USTA2R and USTA3F/USTA4R (Supplementary Table ), respectively, and fused with the geneticin-resistance (GenR) cassette from pFL2 ( ) by double-joint PCR.

    Recombinant:

    Article Title: RNA Chaperone Function of a Universal Stress Protein in Arabidopsis Confers Enhanced Cold Stress Tolerance in Plants
    Article Snippet: .. Recombinant AtUSP or BSA was incubated with ssDNA (M13mp18, NEB), dsDNA (M13mp18 RF I DNA, NEB), and luc mRNA (TriLink Biotechnologies Co., San Diego, CA, USA) in 15 μL binding buffer (10 mM Tris-HCl, pH 7.5) on ice for 30 min. .. The samples were then separated using 0.8% agarose gels, and stained with ethidium bromide to visualize migration shifts.

    Protease Inhibitor:

    Article Title: The Ska complex promotes Aurora B activity to ensure chromosome biorientation
    Article Snippet: .. For the time course assay in , 0.5 µM Aurora B–His6 was preincubated with 0.5 µM BSA, 0.5 µM Ska complex, or 0.5 µM MBP-INCENP790–918 -His6 for 30 min at 30°C in kinase buffer A supplemented with EDTA-free protease inhibitor cocktail before addition of 2 µg histone H3.1 (New England Biolabs, Inc.), 10 µM ATP, and 5 µCi γ-[32 P]ATP. ..

    Generated:

    Article Title: Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System
    Article Snippet: .. A short cassette with two Bsm BI sites was generated by annealing the sense (LINK-SEQ-F) and antisense (LINK-SEQ-R) oligonucleotides (Supplementary Table ) and inserted between the two Bsa I sites of pUC19-tRp-gRNA by Golden Gate cloning (New England Biolabs, Ipswich, MA, United States) as described ( ; ) to generate the pUC19- LINK vector. .. The PGln-tRNA -gRNA cassette flanked by two Bsm BI sites was then released from pUC19-LINK and cloned between the Kpn I and Eco RI sites of pCRISPR/Cas-U6-1 ( ) to generate the pCas9-tRp-gRNA vector.

    Purification:

    Article Title: Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA
    Article Snippet: .. DNase I protection assays Purified plasmid DNA (77 ng) was pre-incubated with CbpA or BSA, at 37°C, in DNase I reaction buffer (NEB) in a final volume of 10 µl. ..

    Incubation:

    Article Title: RNA Chaperone Function of a Universal Stress Protein in Arabidopsis Confers Enhanced Cold Stress Tolerance in Plants
    Article Snippet: .. Recombinant AtUSP or BSA was incubated with ssDNA (M13mp18, NEB), dsDNA (M13mp18 RF I DNA, NEB), and luc mRNA (TriLink Biotechnologies Co., San Diego, CA, USA) in 15 μL binding buffer (10 mM Tris-HCl, pH 7.5) on ice for 30 min. .. The samples were then separated using 0.8% agarose gels, and stained with ethidium bromide to visualize migration shifts.

    Binding Assay:

    Article Title: RNA Chaperone Function of a Universal Stress Protein in Arabidopsis Confers Enhanced Cold Stress Tolerance in Plants
    Article Snippet: .. Recombinant AtUSP or BSA was incubated with ssDNA (M13mp18, NEB), dsDNA (M13mp18 RF I DNA, NEB), and luc mRNA (TriLink Biotechnologies Co., San Diego, CA, USA) in 15 μL binding buffer (10 mM Tris-HCl, pH 7.5) on ice for 30 min. .. The samples were then separated using 0.8% agarose gels, and stained with ethidium bromide to visualize migration shifts.

    Plasmid Preparation:

    Article Title: Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System
    Article Snippet: .. A short cassette with two Bsm BI sites was generated by annealing the sense (LINK-SEQ-F) and antisense (LINK-SEQ-R) oligonucleotides (Supplementary Table ) and inserted between the two Bsa I sites of pUC19-tRp-gRNA by Golden Gate cloning (New England Biolabs, Ipswich, MA, United States) as described ( ; ) to generate the pUC19- LINK vector. .. The PGln-tRNA -gRNA cassette flanked by two Bsm BI sites was then released from pUC19-LINK and cloned between the Kpn I and Eco RI sites of pCRISPR/Cas-U6-1 ( ) to generate the pCas9-tRp-gRNA vector.

    Article Title: Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA
    Article Snippet: .. DNase I protection assays Purified plasmid DNA (77 ng) was pre-incubated with CbpA or BSA, at 37°C, in DNase I reaction buffer (NEB) in a final volume of 10 µl. ..

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    New England Biolabs bsa
    A direct physical interaction between <t>RECQ1</t> and Ku70/80 in vitro . A. RECQ1 directly interacts with Ku70 and Ku80. GST or GST fused-full length RECQ1 was incubated with bacterially expressed Ku70/80, Ku70, Ku80 or N-Ku80 (lacking the C-terminus amino acid residues 565–732) followed by extensive washing of the beads, SDS-PAGE, and Western transfer. The blots were probed separately with anti-His (for Ku detection) and anti-RECQ1 antibodies. Input lanes account for 10% of the bacterial lysate expressing Ku protein used in the pull-down reactions. B. Recombinant RECQ1 and Ku70/80 proteins interact directly as shown by ELISA. Either <t>BSA</t> or purified recombinant Ku70/80 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant RECQ1 (0–50 nM) for 1 h at 30°C. Parallel wells contained DNaseI (100 U/ml) or EtBr (50 µg/ml) in the binding step to test for DNA-mediated protein interaction. Following washing, Ku70/80-bound RECQ1 was detected by ELISA using anti-RECQ1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. C. GST alone or GST-RECQ1 fragments (as indicated) bound to glutathione beads were incubated overnight at 4°C with HeLa extract (500 µg) that was either untreated or pre-treated with benzonase. After extensive washings, the bound Ku70/80 was eluted with SDS sample buffer and analyzed by Western blot using anti-Ku70 and anti-Ku80 antibodies (right). Coomassie staining of the eluted proteins was done to test expression of various GST-fusion fragments of RECQ1 (left). GST-RECQ1 proteins are marked by asterisk. Marker, protein molecular weight marker.
    Bsa, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A direct physical interaction between RECQ1 and Ku70/80 in vitro . A. RECQ1 directly interacts with Ku70 and Ku80. GST or GST fused-full length RECQ1 was incubated with bacterially expressed Ku70/80, Ku70, Ku80 or N-Ku80 (lacking the C-terminus amino acid residues 565–732) followed by extensive washing of the beads, SDS-PAGE, and Western transfer. The blots were probed separately with anti-His (for Ku detection) and anti-RECQ1 antibodies. Input lanes account for 10% of the bacterial lysate expressing Ku protein used in the pull-down reactions. B. Recombinant RECQ1 and Ku70/80 proteins interact directly as shown by ELISA. Either BSA or purified recombinant Ku70/80 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant RECQ1 (0–50 nM) for 1 h at 30°C. Parallel wells contained DNaseI (100 U/ml) or EtBr (50 µg/ml) in the binding step to test for DNA-mediated protein interaction. Following washing, Ku70/80-bound RECQ1 was detected by ELISA using anti-RECQ1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. C. GST alone or GST-RECQ1 fragments (as indicated) bound to glutathione beads were incubated overnight at 4°C with HeLa extract (500 µg) that was either untreated or pre-treated with benzonase. After extensive washings, the bound Ku70/80 was eluted with SDS sample buffer and analyzed by Western blot using anti-Ku70 and anti-Ku80 antibodies (right). Coomassie staining of the eluted proteins was done to test expression of various GST-fusion fragments of RECQ1 (left). GST-RECQ1 proteins are marked by asterisk. Marker, protein molecular weight marker.

    Journal: PLoS ONE

    Article Title: Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    doi: 10.1371/journal.pone.0062481

    Figure Lengend Snippet: A direct physical interaction between RECQ1 and Ku70/80 in vitro . A. RECQ1 directly interacts with Ku70 and Ku80. GST or GST fused-full length RECQ1 was incubated with bacterially expressed Ku70/80, Ku70, Ku80 or N-Ku80 (lacking the C-terminus amino acid residues 565–732) followed by extensive washing of the beads, SDS-PAGE, and Western transfer. The blots were probed separately with anti-His (for Ku detection) and anti-RECQ1 antibodies. Input lanes account for 10% of the bacterial lysate expressing Ku protein used in the pull-down reactions. B. Recombinant RECQ1 and Ku70/80 proteins interact directly as shown by ELISA. Either BSA or purified recombinant Ku70/80 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant RECQ1 (0–50 nM) for 1 h at 30°C. Parallel wells contained DNaseI (100 U/ml) or EtBr (50 µg/ml) in the binding step to test for DNA-mediated protein interaction. Following washing, Ku70/80-bound RECQ1 was detected by ELISA using anti-RECQ1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. C. GST alone or GST-RECQ1 fragments (as indicated) bound to glutathione beads were incubated overnight at 4°C with HeLa extract (500 µg) that was either untreated or pre-treated with benzonase. After extensive washings, the bound Ku70/80 was eluted with SDS sample buffer and analyzed by Western blot using anti-Ku70 and anti-Ku80 antibodies (right). Coomassie staining of the eluted proteins was done to test expression of various GST-fusion fragments of RECQ1 (left). GST-RECQ1 proteins are marked by asterisk. Marker, protein molecular weight marker.

    Article Snippet: T4 Ligase Assay Substrate DNA (100 ng) was mixed in 10 µl of 1x T4 ligase buffer supplemented with 0.2 mg/ml BSA, with indicated amounts of RECQ1 or Ku70/80 protein, pre-incubated for 10 min at room temperature followed by addition of T4 ligase (200 U, NEB) in 10 µl 1x T4 ligase/BSA buffer.

    Techniques: In Vitro, Incubation, SDS Page, Western Blot, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay, Binding Assay, Staining, Marker, Molecular Weight

    SDS-PAGE analysis (with Coomassie blue staining) of in vitro digestions of bovine proteins by recombinant TcoCBc1 and TcoCBc6. Ten micrograms of BSA (A) or bovine IgG (B) was incubated for several hours with 1 μg of recombinant mature proteases

    Journal: Eukaryotic Cell

    Article Title: Molecular and Biochemical Characterization of a Cathepsin B-Like Protease Family Unique to Trypanosoma congolense ▿ ▿ †

    doi: 10.1128/EC.00405-07

    Figure Lengend Snippet: SDS-PAGE analysis (with Coomassie blue staining) of in vitro digestions of bovine proteins by recombinant TcoCBc1 and TcoCBc6. Ten micrograms of BSA (A) or bovine IgG (B) was incubated for several hours with 1 μg of recombinant mature proteases

    Article Snippet: Ten micrograms of purified bovine IgG (Sigma) or bovine serum albumin (BSA) (New England Biolabs) was incubated with 1 μg purified protease in 100 mM Na-acetate buffer, pH 4.5, containing 5 mM dithiothreitol (DTT) for up to 5 h at 37°C.

    Techniques: SDS Page, Staining, In Vitro, Recombinant, Incubation

    Removal of the inhibitory effects of melanin on PCR amplification. (A) Increasing concentrations of synthetic melanin were added to DNA extracted from 1676 melanoma cell lines. (B) Increasing concentrations of BSA (ng/μl) were added to DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. (C) The effect of diluting DNA assessed in the presence of 40 ng/μl of melanin and either 1U or 2U of Taq polymerase. (D) NucleoSpin ® gDNA Clean-up XS Kit used on DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. PCR amplification of the DNA was monitored on 2% gel agarose electrophoresis with ethidium bromide staining. MW, molecular weight markers.

    Journal: PLoS ONE

    Article Title: Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens

    doi: 10.1371/journal.pone.0158698

    Figure Lengend Snippet: Removal of the inhibitory effects of melanin on PCR amplification. (A) Increasing concentrations of synthetic melanin were added to DNA extracted from 1676 melanoma cell lines. (B) Increasing concentrations of BSA (ng/μl) were added to DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. (C) The effect of diluting DNA assessed in the presence of 40 ng/μl of melanin and either 1U or 2U of Taq polymerase. (D) NucleoSpin ® gDNA Clean-up XS Kit used on DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. PCR amplification of the DNA was monitored on 2% gel agarose electrophoresis with ethidium bromide staining. MW, molecular weight markers.

    Article Snippet: To circumvent PCR inhibition, bovine serum albumin (BSA) from New England BioLabs (Hitchin, UK) and the NucleoSpin® gDNA Clean-up XS Kit (Macherey-Nagel, Betheleham, PA, USA) were used.

    Techniques: Polymerase Chain Reaction, Amplification, Cell Culture, Electrophoresis, Staining, Molecular Weight

    Functional and physical interactions between RPA and xDNA2. (A) The effect of RPA on xDNA2’s 5′→3′ exonuclease activity against two different single-stranded oligonucleotides. The substrates were labeled with 32 P-labeled dA (marked by the asterisks) and attached to Streptavidin paramagnetic beads via the 3′ biotin-dC. After incubation at room temperature for 1 h, the reactions were stopped with SDS-EDTA, boiled for 10 min, and separated on a 10% TAE-PAGE. The percentage of the substrate undegraded was relative to the total signal for each reaction. The sizes of the products were determined by separating on a sequencing gel (not depicted). (B) The effect of RPA and T4 gp32 on the nuclease activity of xDNA2. The substrate, 48mer-1 beads, was incubated with various proteins as indicated at room temperature for 1 h and analyzed similarly to that in A. (C) Coimmunoprecipitation of RPA and xDNA2. The immunoprecipitates were separated on an 8% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and probed for different proteins by Western blotting. For RPA, a rat antibody against the p70 subunit was used for Western blotting. Untreated cytosol was loaded at the indicated amounts to provide the standard for quantitation. White lines indicate that intervening lanes have been spliced out. (D) Interaction between the purified RPA and xDNA2. FLAG beads were precoated with either recombinant xDNA2 or BSA and then incubated with the purified RPA protein. The beads and supernatant fractions were analyzed similarly to that in C. xRPA, Xenopus RPA. Ab, antibody.

    Journal: The Journal of Cell Biology

    Article Title: Replication protein A promotes 5?- > 3? end processing during homology-dependent DNA double-strand break repair

    doi: 10.1083/jcb.201005110

    Figure Lengend Snippet: Functional and physical interactions between RPA and xDNA2. (A) The effect of RPA on xDNA2’s 5′→3′ exonuclease activity against two different single-stranded oligonucleotides. The substrates were labeled with 32 P-labeled dA (marked by the asterisks) and attached to Streptavidin paramagnetic beads via the 3′ biotin-dC. After incubation at room temperature for 1 h, the reactions were stopped with SDS-EDTA, boiled for 10 min, and separated on a 10% TAE-PAGE. The percentage of the substrate undegraded was relative to the total signal for each reaction. The sizes of the products were determined by separating on a sequencing gel (not depicted). (B) The effect of RPA and T4 gp32 on the nuclease activity of xDNA2. The substrate, 48mer-1 beads, was incubated with various proteins as indicated at room temperature for 1 h and analyzed similarly to that in A. (C) Coimmunoprecipitation of RPA and xDNA2. The immunoprecipitates were separated on an 8% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and probed for different proteins by Western blotting. For RPA, a rat antibody against the p70 subunit was used for Western blotting. Untreated cytosol was loaded at the indicated amounts to provide the standard for quantitation. White lines indicate that intervening lanes have been spliced out. (D) Interaction between the purified RPA and xDNA2. FLAG beads were precoated with either recombinant xDNA2 or BSA and then incubated with the purified RPA protein. The beads and supernatant fractions were analyzed similarly to that in C. xRPA, Xenopus RPA. Ab, antibody.

    Article Snippet: 50 µl of the peak heparin fraction containing recombinant xDNA2 or 5 µg BSA (New England Biolabs, Inc.) was coated onto 10 µl anti-FLAG M2 agarose beads (Sigma-Aldrich).

    Techniques: Functional Assay, Recombinase Polymerase Amplification, Activity Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Sequencing, SDS Page, Western Blot, Quantitation Assay, Purification, Recombinant