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    Structured Review

    New England Biolabs bsa
    Diagram of the modified <t>Gln-tRNA-gRNA</t> 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 <t>Bsa</t> 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.
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    Images

    1) 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

    2) Product Images from "Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha"

    Article Title: Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha

    Journal: bioRxiv

    doi: 10.1101/2020.02.29.971002

    Key elements in the OpenPlant Loop assembly toolkit. (A) The Phytobrick common syntax defines ten DNA part positions and 12 DNA part fusion sites. For Marchantia, we commonly use eight positions and nine fusion sites, by combining positions A2-A3 for proximal promoter (PROMP), and B1-B2 for 5’ untranslated region (5UTR). The other types of parts are: A1 for distal promoter (PROMD), B3 for coding sequence with start codon and no stop codon (CDS1), B4 for coding sequence without start or stop codon (CDS2), B5 coding sequence without start codon and with stop codon (CTAG), B6 for 3’ untranslated region (3UTR) and C1 for transcription terminator (TERM). Parts can span multiple fusion sites, like A1-A3 for promoter (PROM), A1-B2 for promoter with 5’ UTR (PROM5), B3-B4 for coding sequence with start codon and no stop codon for N-terminal fusion with CTAG (CDS12), B3-B6 for coding sequence with start and stop codons (CDS), or B6-C1 for 3’ UTR with terminator (3TERM). (B) Schematic representation of the pUAP4 vector with divergent Sap I sites to accept L0 parts and convergent Bsa I sites to assemble L0 parts into transcription units (L1). (C) Summary of the Loop acceptor vectors of the OpenPlant toolkit. For nuclear genome transformation: pCk (1,2,3,4) can be used for assembly of L0 parts into a Level 1 plasmid using Bsa I, and pCs (A,B,C,E) can be used for assembly of up to four Level 1 plasmids into a Level 2 construct using Sap I. The vectors pCkchlo (1,2,3,4) and pCschlo (A,B,C,E) can be used for chloroplast applications. The vectors L1_lacZgRNA-Ck2, L1_lacZgRNA-Ck3 and L2_lacZgRNA-Cas9 are designed for CRISPR/Cas9 genome editing. LB and RB: left and right border repeats respectively from nopaline C58 T-DNA. Filled blue rounded rectangle: lacZα cassette for blue-white screening. Filled black circles: pSa origin or replication.
    Figure Legend Snippet: Key elements in the OpenPlant Loop assembly toolkit. (A) The Phytobrick common syntax defines ten DNA part positions and 12 DNA part fusion sites. For Marchantia, we commonly use eight positions and nine fusion sites, by combining positions A2-A3 for proximal promoter (PROMP), and B1-B2 for 5’ untranslated region (5UTR). The other types of parts are: A1 for distal promoter (PROMD), B3 for coding sequence with start codon and no stop codon (CDS1), B4 for coding sequence without start or stop codon (CDS2), B5 coding sequence without start codon and with stop codon (CTAG), B6 for 3’ untranslated region (3UTR) and C1 for transcription terminator (TERM). Parts can span multiple fusion sites, like A1-A3 for promoter (PROM), A1-B2 for promoter with 5’ UTR (PROM5), B3-B4 for coding sequence with start codon and no stop codon for N-terminal fusion with CTAG (CDS12), B3-B6 for coding sequence with start and stop codons (CDS), or B6-C1 for 3’ UTR with terminator (3TERM). (B) Schematic representation of the pUAP4 vector with divergent Sap I sites to accept L0 parts and convergent Bsa I sites to assemble L0 parts into transcription units (L1). (C) Summary of the Loop acceptor vectors of the OpenPlant toolkit. For nuclear genome transformation: pCk (1,2,3,4) can be used for assembly of L0 parts into a Level 1 plasmid using Bsa I, and pCs (A,B,C,E) can be used for assembly of up to four Level 1 plasmids into a Level 2 construct using Sap I. The vectors pCkchlo (1,2,3,4) and pCschlo (A,B,C,E) can be used for chloroplast applications. The vectors L1_lacZgRNA-Ck2, L1_lacZgRNA-Ck3 and L2_lacZgRNA-Cas9 are designed for CRISPR/Cas9 genome editing. LB and RB: left and right border repeats respectively from nopaline C58 T-DNA. Filled blue rounded rectangle: lacZα cassette for blue-white screening. Filled black circles: pSa origin or replication.

    Techniques Used: Sequencing, Plasmid Preparation, Transformation Assay, Construct, CRISPR

    3) 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

    4) Product Images from "Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination"

    Article Title: Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200304154

    LN-2 activates ILK activity and DN-ILK inhibits oligodendrocyte cell spreading. (A) OPCs were transfected with 10 MOI vector alone as a control or with DN-ILK (kinase-inactive form of ILK) by adenovirus gene transfer. After 24 h, transfectants were plated on LN-2 in serum-free media with N 2 and cultured for the indicated time periods. ILK was immunoprecipitated from cell extracts. ILK activity was determined with MBP as an exogenous substrate. Total amounts of ILK proteins in the immunoprecipitates were detected by immunoblot with an anti-ILK antibody. Results indicate that LN-2 stimulates ILK activity. (B) 1 h after cells were plated on BSA, LN-2, TSP-1, and TN-C. ILK activity was detected by phosphorylation of MBP. ILK levels were detected as controls. LN-2 stimulates ILK activity more effectively than any of the other ECMs at 1h (P
    Figure Legend Snippet: LN-2 activates ILK activity and DN-ILK inhibits oligodendrocyte cell spreading. (A) OPCs were transfected with 10 MOI vector alone as a control or with DN-ILK (kinase-inactive form of ILK) by adenovirus gene transfer. After 24 h, transfectants were plated on LN-2 in serum-free media with N 2 and cultured for the indicated time periods. ILK was immunoprecipitated from cell extracts. ILK activity was determined with MBP as an exogenous substrate. Total amounts of ILK proteins in the immunoprecipitates were detected by immunoblot with an anti-ILK antibody. Results indicate that LN-2 stimulates ILK activity. (B) 1 h after cells were plated on BSA, LN-2, TSP-1, and TN-C. ILK activity was detected by phosphorylation of MBP. ILK levels were detected as controls. LN-2 stimulates ILK activity more effectively than any of the other ECMs at 1h (P

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Cell Culture, Immunoprecipitation

    LN-2 promotes oligodendrocyte cell spreading in vitro. (A) Purified OPCs isolated from the rat forebrain were cultured in the presence of PDGF and basic FGF for 3 d. Cells were fixed after three further days of growth on BSA, LN-2, TSP-1, and TN-C in serum-free media with N 2 supplement (N 2 ). Cells were plated and stained for O1 (left, green) and DAPI (blue). Bar, 100 μm. (B) The relative percentages of O1 + cells with broad myelin membrane sheets on different ECM substrates were scored. LN-2 increases the ratio of O1 + cells with myelin membrane sheets to the total number of O1 + cells. Results are presented as ±SEM; and comparisons by ANOVA are significant at P
    Figure Legend Snippet: LN-2 promotes oligodendrocyte cell spreading in vitro. (A) Purified OPCs isolated from the rat forebrain were cultured in the presence of PDGF and basic FGF for 3 d. Cells were fixed after three further days of growth on BSA, LN-2, TSP-1, and TN-C in serum-free media with N 2 supplement (N 2 ). Cells were plated and stained for O1 (left, green) and DAPI (blue). Bar, 100 μm. (B) The relative percentages of O1 + cells with broad myelin membrane sheets on different ECM substrates were scored. LN-2 increases the ratio of O1 + cells with myelin membrane sheets to the total number of O1 + cells. Results are presented as ±SEM; and comparisons by ANOVA are significant at P

    Techniques Used: In Vitro, Purification, Isolation, Cell Culture, Staining

    Oligodendrocyte cell spreading promoted by LN-2 requires PI3K, not MAPK. (A) 45 min and 6 h after cells were plated on BSA, LN-2, TSP-1 and TN-C, phosphorylation of AKT was detected by immunoblot with AKT phospho-ser473 (pAKT) antibody. AKT levels were detected as controls. (B–D) The blocking effects of both 0.01 μM wortmannin (wort) and 0.5 μM LY294002 (PI3K inhibitors) on pAKT or of 4 μM U0126 (MAPK/ERK inhibitor) on pMAPK at 15 and 45 min were shown by immunoblot. AKT or MAPK levels were detected as controls. (E) Cells were fixed after 3 d growth on LN-2 with various inhibitors at the same concentrations used in B–D, after pretreatment for 5 min before plating and staining by O1 (green) and DAPI (blue). Bar, 100 μm. (F) OPCs were plated on LN-2 and infected with GFP-vector (20 MOI) alone or a DN-AKT (10 MOI) in serum-free media with N 2 for 3 d by an adenovirus-mediated tetracycline (Tet)–off inducible system. Double staining with O1 (left, red) and GFP (left, green) or with O1 (middle and right, green) and HA-epitope–tagged DN-AKT (middle and right, red) delineates cells infected by vector or DN-AKT. Incubation of tetracycline responsive promoter (TRE)-DN-AKT (10 MOI) with tetracycline-controlled transactivator (tTA, 10 MOI) blocks LN-2–induced cell spreading by expression of DN-AKT. Incubation with 10 μg/ml Tet turned off the expression of DN-AKT, showing cell spreading as much as GFP-vector alone. Bar, 100 μm. (G) The relative percentage of O1 + cells with broad myelin membrane sheets on LN-2 with various inhibitors is shown. Treatment with PI3K inhibitors reduces myelin membrane formation up to 50% (P
    Figure Legend Snippet: Oligodendrocyte cell spreading promoted by LN-2 requires PI3K, not MAPK. (A) 45 min and 6 h after cells were plated on BSA, LN-2, TSP-1 and TN-C, phosphorylation of AKT was detected by immunoblot with AKT phospho-ser473 (pAKT) antibody. AKT levels were detected as controls. (B–D) The blocking effects of both 0.01 μM wortmannin (wort) and 0.5 μM LY294002 (PI3K inhibitors) on pAKT or of 4 μM U0126 (MAPK/ERK inhibitor) on pMAPK at 15 and 45 min were shown by immunoblot. AKT or MAPK levels were detected as controls. (E) Cells were fixed after 3 d growth on LN-2 with various inhibitors at the same concentrations used in B–D, after pretreatment for 5 min before plating and staining by O1 (green) and DAPI (blue). Bar, 100 μm. (F) OPCs were plated on LN-2 and infected with GFP-vector (20 MOI) alone or a DN-AKT (10 MOI) in serum-free media with N 2 for 3 d by an adenovirus-mediated tetracycline (Tet)–off inducible system. Double staining with O1 (left, red) and GFP (left, green) or with O1 (middle and right, green) and HA-epitope–tagged DN-AKT (middle and right, red) delineates cells infected by vector or DN-AKT. Incubation of tetracycline responsive promoter (TRE)-DN-AKT (10 MOI) with tetracycline-controlled transactivator (tTA, 10 MOI) blocks LN-2–induced cell spreading by expression of DN-AKT. Incubation with 10 μg/ml Tet turned off the expression of DN-AKT, showing cell spreading as much as GFP-vector alone. Bar, 100 μm. (G) The relative percentage of O1 + cells with broad myelin membrane sheets on LN-2 with various inhibitors is shown. Treatment with PI3K inhibitors reduces myelin membrane formation up to 50% (P

    Techniques Used: Blocking Assay, Staining, Infection, Plasmid Preparation, Double Staining, Incubation, Expressing

    5) Product Images from "Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase"

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl060

    Size-exclusion chromatographic analysis of wild-type and S326C OGG1. ( A ) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min ( B ). Identical runs were performed with 100 µg polymorphic S326C OGG1 ( C ) or 100 µg of both wild-type and S326C OGG1 together ( D ).
    Figure Legend Snippet: Size-exclusion chromatographic analysis of wild-type and S326C OGG1. ( A ) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min ( B ). Identical runs were performed with 100 µg polymorphic S326C OGG1 ( C ) or 100 µg of both wild-type and S326C OGG1 together ( D ).

    Techniques Used: Purification, Flow Cytometry

    6) Product Images from "High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection"

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018900

    Protein-DNA interaction analysis based on IFP fusions. ( A ) Fusion proteins ANAC042-TEV-IFP-6xHis (70 kDa) and IFP-6xHis (36 kDa) affinity-purified from E. coli . Two elution fractions (2×250 µL) containing the purified proteins were pooled and analysed after SDS-PAGE by in-gel detection (top) and Coomassie staining (bottom) (lanes 10–13: ANAC042-TEV-IFP-6xHis, 5, 10, 15, and 20 µL; lanes 6 - 9: IFP-6xHis, 2, 5, 8 and 10 µL). BSA served as standard to estimate protein amounts (lanes 1–5: 100/250/500/750/1000 ng). Equal amounts of both proteins (∼5 µg) were used for protein-DNA interaction analysis. M, molecular mass marker (kDa). ( B ) Biotinylated dsDNA was immobilized on streptavidin mutein particles and incubated with ANAC042-TEV-IFP-6xHis protein. After elution, fractions were scanned at 700 nm in the wells of a microtiter plate (strong infrared signal appears white in the digital image). A1: IFP-6xHis input. A2: ANAC042-TEV-IFP-6xHis input. A3: negative control; B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis in the presence of non-biotinylated 7%-DNA. A4: negative control; B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. B1/2: empty wells. B3/4: experiments with B-100%-DNA and B-7%-DNA immobilized on streptavidin mutein beads + ANAC042-TEV-IFP-6xHis incubated in the presence of non-biotinylated 7%- and 100%-DNA, respectively. Areas of the infrared signals were marked (white circles) and integrated signal intensities were calculated (B3 = 319, and B4 = 50). ( C ) After infrared-scanning in microtiter plates (see B) samples were separated by SDS-PAGE and scanned at 700 nm (top) followed by western blot analysis (bottom). Lane 1: IFP-6xHis input (white square). Lane 2: ANAC042-TEV-IFP-6xHis input (white square). Lane 3: negative control with B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 4: experiment with B-100%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 5: negative control with B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. Lane 6: experiment with B-7%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 100%-DNA.
    Figure Legend Snippet: Protein-DNA interaction analysis based on IFP fusions. ( A ) Fusion proteins ANAC042-TEV-IFP-6xHis (70 kDa) and IFP-6xHis (36 kDa) affinity-purified from E. coli . Two elution fractions (2×250 µL) containing the purified proteins were pooled and analysed after SDS-PAGE by in-gel detection (top) and Coomassie staining (bottom) (lanes 10–13: ANAC042-TEV-IFP-6xHis, 5, 10, 15, and 20 µL; lanes 6 - 9: IFP-6xHis, 2, 5, 8 and 10 µL). BSA served as standard to estimate protein amounts (lanes 1–5: 100/250/500/750/1000 ng). Equal amounts of both proteins (∼5 µg) were used for protein-DNA interaction analysis. M, molecular mass marker (kDa). ( B ) Biotinylated dsDNA was immobilized on streptavidin mutein particles and incubated with ANAC042-TEV-IFP-6xHis protein. After elution, fractions were scanned at 700 nm in the wells of a microtiter plate (strong infrared signal appears white in the digital image). A1: IFP-6xHis input. A2: ANAC042-TEV-IFP-6xHis input. A3: negative control; B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis in the presence of non-biotinylated 7%-DNA. A4: negative control; B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. B1/2: empty wells. B3/4: experiments with B-100%-DNA and B-7%-DNA immobilized on streptavidin mutein beads + ANAC042-TEV-IFP-6xHis incubated in the presence of non-biotinylated 7%- and 100%-DNA, respectively. Areas of the infrared signals were marked (white circles) and integrated signal intensities were calculated (B3 = 319, and B4 = 50). ( C ) After infrared-scanning in microtiter plates (see B) samples were separated by SDS-PAGE and scanned at 700 nm (top) followed by western blot analysis (bottom). Lane 1: IFP-6xHis input (white square). Lane 2: ANAC042-TEV-IFP-6xHis input (white square). Lane 3: negative control with B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 4: experiment with B-100%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 5: negative control with B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. Lane 6: experiment with B-7%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 100%-DNA.

    Techniques Used: Affinity Purification, Purification, SDS Page, Staining, Marker, Incubation, Negative Control, Western Blot

    7) Product Images from "Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch"

    Article Title: Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch

    Journal: Nucleic Acids Research

    doi:

    Stimulation of Myh binding to non-cleavable T/GO substrate by Ape1 and H309N. The binding reactions were carried out at 37°C for 10 min with 50 pM T/GO DNA in the presence of Myh, Myh/Ape1, Myh/H309N or Myh/BSA at the concentrations indicated. The binding products were analyzed by EMSA on an 8% native polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left.
    Figure Legend Snippet: Stimulation of Myh binding to non-cleavable T/GO substrate by Ape1 and H309N. The binding reactions were carried out at 37°C for 10 min with 50 pM T/GO DNA in the presence of Myh, Myh/Ape1, Myh/H309N or Myh/BSA at the concentrations indicated. The binding products were analyzed by EMSA on an 8% native polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left.

    Techniques Used: Binding Assay, Labeling

    Stimulation of Myh–DNA complex formation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 10 min with 0.2 nM A/GO substrate and 0.5 nM Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. One aliquot of each reaction was directly analyzed by EMSA on an 8% native polyacrylamide gel. The other aliquot, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. ( A ) Phosphoimage of Myh–DNA complexes. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left. ( B ) Phosphoimage of accumulation of glycosylase product. The intact 96mer DNA (Intact) and cleaved 60mer product (Cleaved product) are indicated on the left.
    Figure Legend Snippet: Stimulation of Myh–DNA complex formation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 10 min with 0.2 nM A/GO substrate and 0.5 nM Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. One aliquot of each reaction was directly analyzed by EMSA on an 8% native polyacrylamide gel. The other aliquot, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. ( A ) Phosphoimage of Myh–DNA complexes. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left. ( B ) Phosphoimage of accumulation of glycosylase product. The intact 96mer DNA (Intact) and cleaved 60mer product (Cleaved product) are indicated on the left.

    Techniques Used: Mutagenesis, Labeling

    Glycosylase stimulation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO substrate and increasing concentrations of Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. Then each reaction mixture, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. A plot of the fraction of glycosylase product versus [Myh] is shown.
    Figure Legend Snippet: Glycosylase stimulation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO substrate and increasing concentrations of Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. Then each reaction mixture, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. A plot of the fraction of glycosylase product versus [Myh] is shown.

    Techniques Used: Mutagenesis

    8) Product Images from "PIE-1 Translation in the Germline Lineage Contributes to PIE-1 Asymmetry in the Early Caenorhabditis elegans Embryo"

    Article Title: PIE-1 Translation in the Germline Lineage Contributes to PIE-1 Asymmetry in the Early Caenorhabditis elegans Embryo

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.118.200744

    Quantification of the increase in PIE-1::GFP concentration in the P lineage. A. Schematic of PIE-1 (gray) localization from the 1-cell to the 4-cell stage. Maternally deposited PIE-1 segregates asymmetrically to the germline blastomeres P1 and P2 during the first two rounds of cell division. PIE-1 is also degraded in somatic cells. Sister cells are connected by a line. B. Top panel: Coomassie stained SDS-PAGE gel of recombinant GFP and BSA, which was used as a loading standard. Bottom panels: Images of N2 and PIE-1::GFP embryos bathed in 300 nM GFP. Images were pseudocolored using the CyanHot lookup table in ImageJ (scale at the bottom). In order to highlight the dimmer PIE-1::GFP signals, the nuclear signal in the main image of the 4-cell embryo is saturated. The image normalization was adjusted equivalently in the 2 and 4-cell embryo insets such that the nuclear signal is not saturated. PIE-1::GFP concentration in the 1-cell embryo was determined using a 150 nM GFP bath, but is shown in a bath of 300 nM GFP to allow comparison with the later stage embryos. C. Estimates of PIE-1::GFP concentration in P0, P1 and P2. For P1 and P2, concentration estimates are shown for the entire cell (Tot), the cytoplasm (Cyt) and for the nucleus (Nuc). Mean concentrations and the number of embryos analyzed are indicated below the graph. Error bars represent 95% confidence intervals. Statistical significance was determined using unpaired t -tests with Welch’s correction for comparisons between embryos (P0 vs. P1; P1 vs. P2) and using paired t -tests for comparisons between cytoplasmic and nuclear concentrations in either P1 or P2. In this and subsequent figures: * = P
    Figure Legend Snippet: Quantification of the increase in PIE-1::GFP concentration in the P lineage. A. Schematic of PIE-1 (gray) localization from the 1-cell to the 4-cell stage. Maternally deposited PIE-1 segregates asymmetrically to the germline blastomeres P1 and P2 during the first two rounds of cell division. PIE-1 is also degraded in somatic cells. Sister cells are connected by a line. B. Top panel: Coomassie stained SDS-PAGE gel of recombinant GFP and BSA, which was used as a loading standard. Bottom panels: Images of N2 and PIE-1::GFP embryos bathed in 300 nM GFP. Images were pseudocolored using the CyanHot lookup table in ImageJ (scale at the bottom). In order to highlight the dimmer PIE-1::GFP signals, the nuclear signal in the main image of the 4-cell embryo is saturated. The image normalization was adjusted equivalently in the 2 and 4-cell embryo insets such that the nuclear signal is not saturated. PIE-1::GFP concentration in the 1-cell embryo was determined using a 150 nM GFP bath, but is shown in a bath of 300 nM GFP to allow comparison with the later stage embryos. C. Estimates of PIE-1::GFP concentration in P0, P1 and P2. For P1 and P2, concentration estimates are shown for the entire cell (Tot), the cytoplasm (Cyt) and for the nucleus (Nuc). Mean concentrations and the number of embryos analyzed are indicated below the graph. Error bars represent 95% confidence intervals. Statistical significance was determined using unpaired t -tests with Welch’s correction for comparisons between embryos (P0 vs. P1; P1 vs. P2) and using paired t -tests for comparisons between cytoplasmic and nuclear concentrations in either P1 or P2. In this and subsequent figures: * = P

    Techniques Used: Concentration Assay, Staining, SDS Page, Recombinant

    9) Product Images from "Single-domain antibody screening by isPLA-seq"

    Article Title: Single-domain antibody screening by isPLA-seq

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202101115

    High-sensitive, high-throughput screening for anti-SQSTM1 sdAbs by in situ proximity ligation assay ( is PLA)-seq. (A) Workflow of sdAb library screening by is PLA-seq. A Flag-tagged sdAb library containing a randomized 21-aa CDR3 domain with C-terminal 3× Flag tag was synthesized and transiently co-transfected with HA-tagged bait into HEK293T cells. At 48 h after transfection, cells were fixed and probed by is PLA. The is PLA-positive cells were sorted by FACS followed by CDR3 DNA amplification. The recovered cDNAs can be reconstructed for another round screening until the final sequencing and functional validation. The CDR3-null vector was used as a sdAb control (Con). (B) Flow cytometric analysis of is PLA-positive cells compared with the sdAb control in HEK293T cells ( is PLA control). (C) Representative positive is PLA signals (red dots) in HEK293T cells, compared with the control of anti-HA antibody alone. Nuclei are counterstained with DAPI (blue). Scale bar, 10 μm. (D) PCR products of the recovered is PLA positive HEK293T cells were evaluated by agarose gel electrophoresis. sdAb Con vector, non-transfected HEK3293T cells, is PLA positive HEK293T cells, and H 2 O were used as templates, respectively. Right lane, DNA markers. bp, base pairs. (E) Recombinant anti-SQSTM1 sdAb was produced in E. coli BL21 cells and was analyzed by SDS–PAGE followed by Coomassie blue staining, with BSA as a loading control. Upper panel, schematic showing the tandem fusion construct containing GST, tobacco etch virus cleavage site, anti-SQSTM1 sdAb or CDR3-null control (sdAb Con), the translocation domain of Pseudomonas exotoxin A (ETA), and 3× Flag tag. WCL, whole BL21 cell lysate with 0.2 mM IPTG induction; B, binding proteins on GSH resin; P, purified sdAb; M, protein molecular markers. Arrow, the purified sdAb clone #1 (upper) or sdAb Con (lower), respectively. Source data are available for this figure.
    Figure Legend Snippet: High-sensitive, high-throughput screening for anti-SQSTM1 sdAbs by in situ proximity ligation assay ( is PLA)-seq. (A) Workflow of sdAb library screening by is PLA-seq. A Flag-tagged sdAb library containing a randomized 21-aa CDR3 domain with C-terminal 3× Flag tag was synthesized and transiently co-transfected with HA-tagged bait into HEK293T cells. At 48 h after transfection, cells were fixed and probed by is PLA. The is PLA-positive cells were sorted by FACS followed by CDR3 DNA amplification. The recovered cDNAs can be reconstructed for another round screening until the final sequencing and functional validation. The CDR3-null vector was used as a sdAb control (Con). (B) Flow cytometric analysis of is PLA-positive cells compared with the sdAb control in HEK293T cells ( is PLA control). (C) Representative positive is PLA signals (red dots) in HEK293T cells, compared with the control of anti-HA antibody alone. Nuclei are counterstained with DAPI (blue). Scale bar, 10 μm. (D) PCR products of the recovered is PLA positive HEK293T cells were evaluated by agarose gel electrophoresis. sdAb Con vector, non-transfected HEK3293T cells, is PLA positive HEK293T cells, and H 2 O were used as templates, respectively. Right lane, DNA markers. bp, base pairs. (E) Recombinant anti-SQSTM1 sdAb was produced in E. coli BL21 cells and was analyzed by SDS–PAGE followed by Coomassie blue staining, with BSA as a loading control. Upper panel, schematic showing the tandem fusion construct containing GST, tobacco etch virus cleavage site, anti-SQSTM1 sdAb or CDR3-null control (sdAb Con), the translocation domain of Pseudomonas exotoxin A (ETA), and 3× Flag tag. WCL, whole BL21 cell lysate with 0.2 mM IPTG induction; B, binding proteins on GSH resin; P, purified sdAb; M, protein molecular markers. Arrow, the purified sdAb clone #1 (upper) or sdAb Con (lower), respectively. Source data are available for this figure.

    Techniques Used: High Throughput Screening Assay, In Situ, Proximity Ligation Assay, Library Screening, FLAG-tag, Synthesized, Transfection, FACS, Amplification, Sequencing, Functional Assay, Plasmid Preparation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Recombinant, Produced, SDS Page, Staining, Construct, Translocation Assay, Binding Assay, Purification

    10) Product Images from "Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks"

    Article Title: Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks

    Journal: Nature cell biology

    doi: 10.1038/ncb3643

    Sequence-specific localization of DDRNAs at DNA damage sites is transcription-dependent. ( A ) Images of NIH2/4 cells expressing GFP-LacR, microinjected with double-stranded DDRNA-Cy5, artificial CXCR4-Cy5 miRNA (Ctrl RNA 1) or let-7a-Cy5 miRNA (Ctrl RNA 2), together with BSA (-) or I-SceI restriction enzyme (+) and imaged 4 h post injection. Scale bar 5 µm. Inset is a magnified view of the boxed region. Images from one out of 3 experiments with similar results. ( B ) Quantification of (A) showing the number of fluorophore-labeled RNA molecules at the locus as measured by single-molecule analysis based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( C ) DDRNAs localize at the damage site to restore DDR focus formation. NIH2/4 cells knocked-down for Dicer and Drosha were mildly permeabilized and incubated with DDRNA-Cy5 or CXCR4-Cy5 (Ctrl RNA 1). The bar plot shows the percentage of cells positive for co-localization of 53BP1 with TetR, of RNA-Cy5 with TetR and the triple co-localization of 53BP1, RNA-Cy5 and TetR. Error bars indicate SEM (for siLuc and siDic n=4, for siDro n=3 independent experiments, ≥70 cells analysed in total per condition). ( D ) NIH2/4 cells expressing YFP-TetR and inducible I-SceI were treated with AM, DRB or ACTD at low and high doses or vehicle alone for 2 h before cut induction, then mildly permeabilized and incubated with DDRNA-Cy5. The bar plots show the percentage of cells in which DDRNA signal co-localizes with the TetR spot. Error bars indicate SEM (n=3 independent experiments, ≥80 cells analysed in total per condition). ( E ) NIH2/4 cells expressing GFP-LacR were microinjected with double-stranded DDRNA-Cy5, together with I-SceI protein and AM and imaged 4 h post injection. The plot shows the number of DDRNA molecules at the locus as measured by single-molecule counting based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( B,E ) P values were calculated using two-tailed t-test. ( C,D ) P values were calculated using chi-squared test. *** P
    Figure Legend Snippet: Sequence-specific localization of DDRNAs at DNA damage sites is transcription-dependent. ( A ) Images of NIH2/4 cells expressing GFP-LacR, microinjected with double-stranded DDRNA-Cy5, artificial CXCR4-Cy5 miRNA (Ctrl RNA 1) or let-7a-Cy5 miRNA (Ctrl RNA 2), together with BSA (-) or I-SceI restriction enzyme (+) and imaged 4 h post injection. Scale bar 5 µm. Inset is a magnified view of the boxed region. Images from one out of 3 experiments with similar results. ( B ) Quantification of (A) showing the number of fluorophore-labeled RNA molecules at the locus as measured by single-molecule analysis based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( C ) DDRNAs localize at the damage site to restore DDR focus formation. NIH2/4 cells knocked-down for Dicer and Drosha were mildly permeabilized and incubated with DDRNA-Cy5 or CXCR4-Cy5 (Ctrl RNA 1). The bar plot shows the percentage of cells positive for co-localization of 53BP1 with TetR, of RNA-Cy5 with TetR and the triple co-localization of 53BP1, RNA-Cy5 and TetR. Error bars indicate SEM (for siLuc and siDic n=4, for siDro n=3 independent experiments, ≥70 cells analysed in total per condition). ( D ) NIH2/4 cells expressing YFP-TetR and inducible I-SceI were treated with AM, DRB or ACTD at low and high doses or vehicle alone for 2 h before cut induction, then mildly permeabilized and incubated with DDRNA-Cy5. The bar plots show the percentage of cells in which DDRNA signal co-localizes with the TetR spot. Error bars indicate SEM (n=3 independent experiments, ≥80 cells analysed in total per condition). ( E ) NIH2/4 cells expressing GFP-LacR were microinjected with double-stranded DDRNA-Cy5, together with I-SceI protein and AM and imaged 4 h post injection. The plot shows the number of DDRNA molecules at the locus as measured by single-molecule counting based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( B,E ) P values were calculated using two-tailed t-test. ( C,D ) P values were calculated using chi-squared test. *** P

    Techniques Used: Sequencing, Expressing, Injection, Labeling, Incubation, Single Molecule Counting, Two Tailed Test

    11) Product Images from "Fic Proteins Inhibit the Activity of Topoisomerase IV by AMPylation in Diverse Bacteria"

    Article Title: Fic Proteins Inhibit the Activity of Topoisomerase IV by AMPylation in Diverse Bacteria

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.02084

    Fic-1 interacts with ParE. (A) Fic-1 interacts with PfParE in a bacterial two-hybrid assay. Fic-1 and ParE were fused to the T25 and T18 fragments of the Bordetella pertussis adenylate cyclase, respectively. The plasmids pKT25-zip and pUT18C-zip, expressing T25 and T18 fused to a leucine zipper motif that strongly interact, respectively, serve as the positive control. Cells containing the indicated plasmids were grown in LB liquid for 24 h at 30°C. The interactions were quantified by measuring galactosidase activity. Data shown represent the average of three independent experiments. p -values were calculated using the Student’s t -test. (B) Fic-1 interacts with ParE in vitro . Fifty micrograms of Fic-1-His 6 or Fic-1 H135A -His 6 was used to coat Affigel-15 beads, control beads were coated with 50 μg BSA. After blocking with 20 mM Tris–HCl, washed beads were incubated with 40 μg ParE for 4 h at 4°C. After extensive washing, bound proteins were separated by SDS-PAGE and detected by Coomassie brilliant blue staining. 10% input was loaded as a reference. Note that the predicted molecular mass of Fic-1-His 6 is about 23.34 kDa, and His 6 -SUMO-PfParE is 83.33 kDa. Data shown are representative of three experiments with similar results.
    Figure Legend Snippet: Fic-1 interacts with ParE. (A) Fic-1 interacts with PfParE in a bacterial two-hybrid assay. Fic-1 and ParE were fused to the T25 and T18 fragments of the Bordetella pertussis adenylate cyclase, respectively. The plasmids pKT25-zip and pUT18C-zip, expressing T25 and T18 fused to a leucine zipper motif that strongly interact, respectively, serve as the positive control. Cells containing the indicated plasmids were grown in LB liquid for 24 h at 30°C. The interactions were quantified by measuring galactosidase activity. Data shown represent the average of three independent experiments. p -values were calculated using the Student’s t -test. (B) Fic-1 interacts with ParE in vitro . Fifty micrograms of Fic-1-His 6 or Fic-1 H135A -His 6 was used to coat Affigel-15 beads, control beads were coated with 50 μg BSA. After blocking with 20 mM Tris–HCl, washed beads were incubated with 40 μg ParE for 4 h at 4°C. After extensive washing, bound proteins were separated by SDS-PAGE and detected by Coomassie brilliant blue staining. 10% input was loaded as a reference. Note that the predicted molecular mass of Fic-1-His 6 is about 23.34 kDa, and His 6 -SUMO-PfParE is 83.33 kDa. Data shown are representative of three experiments with similar results.

    Techniques Used: Two Hybrid Assay, Expressing, Positive Control, Activity Assay, In Vitro, Blocking Assay, Incubation, SDS Page, Staining

    12) Product Images from "Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth ▿"

    Article Title: Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01506-10

    HtpB interacts with mammalian SAMDC. (a) Far-Western blot assay with 5 or 10 μg of immobilized purified proteins (HtpB, GroEL, and BSA) overlaid with a lysate of L929 cells (L). The negative control for the SAMDC immunostaining signal was prepared
    Figure Legend Snippet: HtpB interacts with mammalian SAMDC. (a) Far-Western blot assay with 5 or 10 μg of immobilized purified proteins (HtpB, GroEL, and BSA) overlaid with a lysate of L929 cells (L). The negative control for the SAMDC immunostaining signal was prepared

    Techniques Used: Far Western Blot, Purification, Negative Control, Immunostaining

    13) Product Images from "GT198 Is a Target of Oncology Drugs and Anticancer Herbs"

    Article Title: GT198 Is a Target of Oncology Drugs and Anticancer Herbs

    Journal: Frontiers in oral health

    doi: 10.3389/froh.2021.679460

    GT198 is a target of oncology drugs. (A) A model of allosteric or competitive drug inhibition of the DNA binding of GT198 dimer. (B) A flowchart of the DNA-binding assay. (C) The increasing concentrations of biotinylated DNA bind to GT198 at EC50 = 43 nM using BSA as a negative control. (D) Inhibition of DNA binding to GT198 by increasing concentrations of clinical drugs as indicated. IC 50 s are measured by sigmoidal curve fit. Data represent mean ± s.e.m of duplicate experiments. (E) A graph model comparing binding affinity and efficacy.
    Figure Legend Snippet: GT198 is a target of oncology drugs. (A) A model of allosteric or competitive drug inhibition of the DNA binding of GT198 dimer. (B) A flowchart of the DNA-binding assay. (C) The increasing concentrations of biotinylated DNA bind to GT198 at EC50 = 43 nM using BSA as a negative control. (D) Inhibition of DNA binding to GT198 by increasing concentrations of clinical drugs as indicated. IC 50 s are measured by sigmoidal curve fit. Data represent mean ± s.e.m of duplicate experiments. (E) A graph model comparing binding affinity and efficacy.

    Techniques Used: Inhibition, Binding Assay, DNA Binding Assay, Negative Control

    14) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Mobius Assembly standard part generation. (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.
    Figure Legend Snippet: Mobius Assembly standard part generation. (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.

    Techniques Used: Plasmid Preparation, Clone Assay, Marker, Amplification, Functional Assay, Sequencing

    15) Product Images from "Simultaneous sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level †Electronic supplementary information (ESI) available: Molecular mechanism of the DNA glycosylase-mediated cleavage of molecular beacons and optimization of the experimental conditions. See DOI: 10.1039/c7sc04296e"

    Article Title: Simultaneous sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level †Electronic supplementary information (ESI) available: Molecular mechanism of the DNA glycosylase-mediated cleavage of molecular beacons and optimization of the experimental conditions. See DOI: 10.1039/c7sc04296e

    Journal: Chemical Science

    doi: 10.1039/c7sc04296e

    Measurement of the Cy3 counts and Cy5 counts in response to the reaction buffer (control), 0.1 g L –1 BSA, 0.1 U μL –1 UDG, 0.1 U μL –1 TDG, 0.1 U μL –1 hOGG1, 0.1 U μL –1 hAAG and 0.1 U μL –1 hOGG1 + 0.1 U μL –1 hAAG. The Cy3-labeled molecular beacon (0.3 μM), Cy5-labeled molecular beacon (0.3 μM) and APE1 (0.1 U μL –1 ) were used in this research. The error bars represent the standard deviations of the three experiments.
    Figure Legend Snippet: Measurement of the Cy3 counts and Cy5 counts in response to the reaction buffer (control), 0.1 g L –1 BSA, 0.1 U μL –1 UDG, 0.1 U μL –1 TDG, 0.1 U μL –1 hOGG1, 0.1 U μL –1 hAAG and 0.1 U μL –1 hOGG1 + 0.1 U μL –1 hAAG. The Cy3-labeled molecular beacon (0.3 μM), Cy5-labeled molecular beacon (0.3 μM) and APE1 (0.1 U μL –1 ) were used in this research. The error bars represent the standard deviations of the three experiments.

    Techniques Used: Labeling

    16) 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

    17) Product Images from "Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion"

    Article Title: Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2011.07927.x

    Screening of EHEC O157:H7 O-island (OI) mutants for altered levels of T3S. SDS-PAGE gels showing culture supernatant secretion profiles for EHEC strain TUV93-0 and a selection of 24 isogenic deletion strains. Parent strain TUV93-0 also acted a positive control for T3S and a T3S system mutant (ΔLEE1–3) provided a negative control for secretion. OI deletions are as defined with reference to the original designations in Perna et al . (2001 ). The translocon protein bands are indicated, EspB/D and EspA as well as BSA which was added as a loading control to rule out the precipitation process as a source of variation and to act as a co-precipitant. Strains were cultured in MEM-HEPES to an OD 600 of 1.0 and culture supernatants were TCA-precipitated, separated by SDS-PAGE and stained with Colloidal blue as described in Experimental procedures .
    Figure Legend Snippet: Screening of EHEC O157:H7 O-island (OI) mutants for altered levels of T3S. SDS-PAGE gels showing culture supernatant secretion profiles for EHEC strain TUV93-0 and a selection of 24 isogenic deletion strains. Parent strain TUV93-0 also acted a positive control for T3S and a T3S system mutant (ΔLEE1–3) provided a negative control for secretion. OI deletions are as defined with reference to the original designations in Perna et al . (2001 ). The translocon protein bands are indicated, EspB/D and EspA as well as BSA which was added as a loading control to rule out the precipitation process as a source of variation and to act as a co-precipitant. Strains were cultured in MEM-HEPES to an OD 600 of 1.0 and culture supernatants were TCA-precipitated, separated by SDS-PAGE and stained with Colloidal blue as described in Experimental procedures .

    Techniques Used: SDS Page, Selection, Positive Control, Mutagenesis, Negative Control, Activated Clotting Time Assay, Cell Culture, Staining

    18) Product Images from "A sensitive electrochemical assay for T4 polynucleotide kinase activity based on titanium dioxide nanotubes and a rolling circle amplification strategy †"

    Article Title: A sensitive electrochemical assay for T4 polynucleotide kinase activity based on titanium dioxide nanotubes and a rolling circle amplification strategy †

    Journal: RSC Advances

    doi: 10.1039/c8ra07745b

    Selectivity of the proposed amplification strategy for the T4 PNK assay. Experimental conditions: 15 U mL −1 T4 PNK, 20 U mL −1 inactivated T4 PNK, phi29 and KF, 1 mg mL −1 BSA. The error bars represent the standard deviation of three repetitive measurements.
    Figure Legend Snippet: Selectivity of the proposed amplification strategy for the T4 PNK assay. Experimental conditions: 15 U mL −1 T4 PNK, 20 U mL −1 inactivated T4 PNK, phi29 and KF, 1 mg mL −1 BSA. The error bars represent the standard deviation of three repetitive measurements.

    Techniques Used: Amplification, Standard Deviation

    19) 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

    20) Product Images from "The fragile X mental retardation protein inhibits translation via interacting with mRNA"

    Article Title: The fragile X mental retardation protein inhibits translation via interacting with mRNA

    Journal: Nucleic Acids Research

    doi:

    Dose-dependent reduction of translation of brain poly(A) RNA but not rabbit reticulocyte poly(A) RNA by FMRP. ( A ) Dose-dependent effect of FMRP. An aliquot of 1.5 µg poly(A) RNA was used in each reaction. Translation yield for each reaction is represented by the percentage of TCA precipitable counts (c.p.m.) obtained from mock-treated reactions. At 10 min after initiation of translation yields for reactions exposed to various amounts of FMRP or BSA as depicted at the bottom were subjected to one way ANOVA analysis ( P
    Figure Legend Snippet: Dose-dependent reduction of translation of brain poly(A) RNA but not rabbit reticulocyte poly(A) RNA by FMRP. ( A ) Dose-dependent effect of FMRP. An aliquot of 1.5 µg poly(A) RNA was used in each reaction. Translation yield for each reaction is represented by the percentage of TCA precipitable counts (c.p.m.) obtained from mock-treated reactions. At 10 min after initiation of translation yields for reactions exposed to various amounts of FMRP or BSA as depicted at the bottom were subjected to one way ANOVA analysis ( P

    Techniques Used:

    21) Product Images from "The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1"

    Article Title: The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr040

    The phospho-mutant at S652 of Rph1 increases UV sensitivity and impairs the dissociation after UV irradiation. ( A ) In vitro kinase assay was performed by recombinant Rph1 or BSA incubated with or without V5-IP WT or KD Rad53 supplied by γ 32 P-ATP. The signal was detected by autoradiography. pRad53 indicated the autophosphorylation of Rad53. pRph1 indicated the phosphorylation of Rph1. Coomassie Blue and immnoblotting (anti-V5) showed the loading controls. ( B ) UV sensitivity of rph1Δ cells containing control vector, WT Rph1 ( RPH1 ) or phospho-defective Rph1 mutants. ( C and D ) The indicated strains as in (B) were harvested for RT-qPCR to detect PHR1 expression in response to UV or not (C) and for HA-ChIP to measure the association of Rph1 at URS of PHR1 (D). Error bars show the SD of three biological repeats. * P
    Figure Legend Snippet: The phospho-mutant at S652 of Rph1 increases UV sensitivity and impairs the dissociation after UV irradiation. ( A ) In vitro kinase assay was performed by recombinant Rph1 or BSA incubated with or without V5-IP WT or KD Rad53 supplied by γ 32 P-ATP. The signal was detected by autoradiography. pRad53 indicated the autophosphorylation of Rad53. pRph1 indicated the phosphorylation of Rph1. Coomassie Blue and immnoblotting (anti-V5) showed the loading controls. ( B ) UV sensitivity of rph1Δ cells containing control vector, WT Rph1 ( RPH1 ) or phospho-defective Rph1 mutants. ( C and D ) The indicated strains as in (B) were harvested for RT-qPCR to detect PHR1 expression in response to UV or not (C) and for HA-ChIP to measure the association of Rph1 at URS of PHR1 (D). Error bars show the SD of three biological repeats. * P

    Techniques Used: Mutagenesis, Irradiation, In Vitro, Kinase Assay, Recombinant, Incubation, Autoradiography, Plasmid Preparation, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation

    22) Product Images from "The barrier-to-autointegration protein is a host factor for HIV type 1 integration"

    Article Title: The barrier-to-autointegration protein is a host factor for HIV type 1 integration

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

    doi:

    Functional reconstitution of salt-stripped HIV-1 PICs with uninfected cell extract. Integration reactions were deproteinized and analyzed by Southern blotting. Lane 1, integration activity of untreated, gradient-purified PICs. Lane 2, HIV-1 PICs treated with 1.2 M KCl and then purified by spin column chromatography and gradient centrifugation before the integration assay. Lane 3, BSA included in the reconstitution buffer. Lanes 4–6 included 5, 10, and 15 μl, of crude cell extract, corresponding to approximately 35, 70, and 105 μg of total protein, respectively. cDNA, 9.7-kb HIV-1 linear DNA substrate; IP, 15.1-kb integration product. Approximately 3-fold more material was loaded in lanes 2–6, accounting for the increased level of cDNA.
    Figure Legend Snippet: Functional reconstitution of salt-stripped HIV-1 PICs with uninfected cell extract. Integration reactions were deproteinized and analyzed by Southern blotting. Lane 1, integration activity of untreated, gradient-purified PICs. Lane 2, HIV-1 PICs treated with 1.2 M KCl and then purified by spin column chromatography and gradient centrifugation before the integration assay. Lane 3, BSA included in the reconstitution buffer. Lanes 4–6 included 5, 10, and 15 μl, of crude cell extract, corresponding to approximately 35, 70, and 105 μg of total protein, respectively. cDNA, 9.7-kb HIV-1 linear DNA substrate; IP, 15.1-kb integration product. Approximately 3-fold more material was loaded in lanes 2–6, accounting for the increased level of cDNA.

    Techniques Used: Functional Assay, Southern Blot, Activity Assay, Purification, Column Chromatography, Gradient Centrifugation

    23) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Mobius Assembly standard part generation. (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.
    Figure Legend Snippet: Mobius Assembly standard part generation. (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.

    Techniques Used: Plasmid Preparation, Clone Assay, Marker, Amplification, Functional Assay, Sequencing

    24) Product Images from "A Portable Droplet Magnetofluidic Device for Point-of-Care Detection of Multidrug-Resistant Candida auris"

    Article Title: A Portable Droplet Magnetofluidic Device for Point-of-Care Detection of Multidrug-Resistant Candida auris

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2022.826694

    Development of benchtop droplet magnetofluidic-compatible rapid duplex qPCR. The step-by-step development process shown here employs benchtop extracted and purified DNA (100 fg) from 3 strains of C. auris that originate from 2 major clades (South Asia and Africa) and a control DNA plasmid containing the bicoid gene (10 pg) as the inputs. (A) Using Promega GoTaq Probe qPCR Master Mix, which is compatible with droplet magnetofluidics, under its fast cycling protocol (95°C hot start for 2 min and 40 cycles of 95°C for 3 s and 60°C for 30 s), both C. auris DNA and control DNA plasmid directly spiked into the PCR mix can be detected, as indicated by clear cycle of quantification (C q ) values in both the FAM channel associated with the C. auris probe (green) and the TYE channel associated with the bicoid probe (red). The C q values are comparable among the 3 strains of C. auris . (B) Addition of BSA and Tween 20—standard additives for PCR in droplet magnetofluidic assays—causes negligible differences to the PCR assay. (C) Accelerating the PCR assay by reducing the annealing and extension step at 60°C from 30 to 15 s only slightly delays the C q values. (D) Appending manual magnetic capture and transport of both C. auris DNA and control DNA plasmid upstream to the accelerated rapid duplex qPCR assay still result in successful detection of both targets. This assay can be readily adapted into the droplet magnetofluidic cartridge and device.
    Figure Legend Snippet: Development of benchtop droplet magnetofluidic-compatible rapid duplex qPCR. The step-by-step development process shown here employs benchtop extracted and purified DNA (100 fg) from 3 strains of C. auris that originate from 2 major clades (South Asia and Africa) and a control DNA plasmid containing the bicoid gene (10 pg) as the inputs. (A) Using Promega GoTaq Probe qPCR Master Mix, which is compatible with droplet magnetofluidics, under its fast cycling protocol (95°C hot start for 2 min and 40 cycles of 95°C for 3 s and 60°C for 30 s), both C. auris DNA and control DNA plasmid directly spiked into the PCR mix can be detected, as indicated by clear cycle of quantification (C q ) values in both the FAM channel associated with the C. auris probe (green) and the TYE channel associated with the bicoid probe (red). The C q values are comparable among the 3 strains of C. auris . (B) Addition of BSA and Tween 20—standard additives for PCR in droplet magnetofluidic assays—causes negligible differences to the PCR assay. (C) Accelerating the PCR assay by reducing the annealing and extension step at 60°C from 30 to 15 s only slightly delays the C q values. (D) Appending manual magnetic capture and transport of both C. auris DNA and control DNA plasmid upstream to the accelerated rapid duplex qPCR assay still result in successful detection of both targets. This assay can be readily adapted into the droplet magnetofluidic cartridge and device.

    Techniques Used: Real-time Polymerase Chain Reaction, Purification, Plasmid Preparation, Polymerase Chain Reaction

    25) 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

    26) Product Images from "Fic Proteins Inhibit the Activity of Topoisomerase IV by AMPylation in Diverse Bacteria"

    Article Title: Fic Proteins Inhibit the Activity of Topoisomerase IV by AMPylation in Diverse Bacteria

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.02084

    Fic-1 interacts with ParE. (A) Fic-1 interacts with PfParE in a bacterial two-hybrid assay. Fic-1 and ParE were fused to the T25 and T18 fragments of the Bordetella pertussis adenylate cyclase, respectively. The plasmids pKT25-zip and pUT18C-zip, expressing T25 and T18 fused to a leucine zipper motif that strongly interact, respectively, serve as the positive control. Cells containing the indicated plasmids were grown in LB liquid for 24 h at 30°C. The interactions were quantified by measuring galactosidase activity. Data shown represent the average of three independent experiments. p -values were calculated using the Student’s t -test. (B) Fic-1 interacts with ParE in vitro . Fifty micrograms of Fic-1-His 6 or Fic-1 H135A -His 6 was used to coat Affigel-15 beads, control beads were coated with 50 μg BSA. After blocking with 20 mM Tris–HCl, washed beads were incubated with 40 μg ParE for 4 h at 4°C. After extensive washing, bound proteins were separated by SDS-PAGE and detected by Coomassie brilliant blue staining. 10% input was loaded as a reference. Note that the predicted molecular mass of Fic-1-His 6 is about 23.34 kDa, and His 6 -SUMO-PfParE is 83.33 kDa. Data shown are representative of three experiments with similar results.
    Figure Legend Snippet: Fic-1 interacts with ParE. (A) Fic-1 interacts with PfParE in a bacterial two-hybrid assay. Fic-1 and ParE were fused to the T25 and T18 fragments of the Bordetella pertussis adenylate cyclase, respectively. The plasmids pKT25-zip and pUT18C-zip, expressing T25 and T18 fused to a leucine zipper motif that strongly interact, respectively, serve as the positive control. Cells containing the indicated plasmids were grown in LB liquid for 24 h at 30°C. The interactions were quantified by measuring galactosidase activity. Data shown represent the average of three independent experiments. p -values were calculated using the Student’s t -test. (B) Fic-1 interacts with ParE in vitro . Fifty micrograms of Fic-1-His 6 or Fic-1 H135A -His 6 was used to coat Affigel-15 beads, control beads were coated with 50 μg BSA. After blocking with 20 mM Tris–HCl, washed beads were incubated with 40 μg ParE for 4 h at 4°C. After extensive washing, bound proteins were separated by SDS-PAGE and detected by Coomassie brilliant blue staining. 10% input was loaded as a reference. Note that the predicted molecular mass of Fic-1-His 6 is about 23.34 kDa, and His 6 -SUMO-PfParE is 83.33 kDa. Data shown are representative of three experiments with similar results.

    Techniques Used: Two Hybrid Assay, Expressing, Positive Control, Activity Assay, In Vitro, Blocking Assay, Incubation, SDS Page, Staining

    27) Product Images from "Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies"

    Article Title: Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18050937

    Chitin-binding activity of recombinant Cts1. ( A ) SDS-PAGE analysis of fractions obtained from IMAC purification of His-tagged Cts1 (Cts1 H ) produced in Escherichia coli . CE, cell extract; FT, flow through; W, wash step; E, elution fractions using different concentrations of imidazole (subscripts). Cts1 H is depicted with a black arrowhead; ( B ) Chitin activity assay with distinct amounts of IMAC purified Cts1 H . Activity was determined by monitoring the change in fluorescence using the fluorogenic chitinase substrate 4-methylumbelliferyl β- d - N , N ′, N ′′-triacetylchitotrioside (4-MUC). RFU, relative fluorescence units; ( C ) Chitin-binding activity of distinct amounts (5 or 10 µg) of purified Cts1 H (indicated by black arrowhead). Equal amounts of purified BSA (NEB, Ipswich, MA, USA) were used as a negative control (depicted by open arrowhead). Chitin Magnetic Beads were mixed with respective proteins (IN), washed rigorously and bound protein was eluted using Laemmli buffer (OUT).
    Figure Legend Snippet: Chitin-binding activity of recombinant Cts1. ( A ) SDS-PAGE analysis of fractions obtained from IMAC purification of His-tagged Cts1 (Cts1 H ) produced in Escherichia coli . CE, cell extract; FT, flow through; W, wash step; E, elution fractions using different concentrations of imidazole (subscripts). Cts1 H is depicted with a black arrowhead; ( B ) Chitin activity assay with distinct amounts of IMAC purified Cts1 H . Activity was determined by monitoring the change in fluorescence using the fluorogenic chitinase substrate 4-methylumbelliferyl β- d - N , N ′, N ′′-triacetylchitotrioside (4-MUC). RFU, relative fluorescence units; ( C ) Chitin-binding activity of distinct amounts (5 or 10 µg) of purified Cts1 H (indicated by black arrowhead). Equal amounts of purified BSA (NEB, Ipswich, MA, USA) were used as a negative control (depicted by open arrowhead). Chitin Magnetic Beads were mixed with respective proteins (IN), washed rigorously and bound protein was eluted using Laemmli buffer (OUT).

    Techniques Used: Binding Assay, Activity Assay, Recombinant, SDS Page, Purification, Produced, Flow Cytometry, Fluorescence, Negative Control, Magnetic Beads

    28) 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

    29) Product Images from "Replication protein A promotes 5?- > 3? end processing during homology-dependent DNA double-strand break repair"

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

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201005110

    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.
    Figure Legend 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.

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

    30) Product Images from "Repair of Gaps in Retroviral DNA Integration Intermediates"

    Article Title: Repair of Gaps in Retroviral DNA Integration Intermediates

    Journal: Journal of Virology

    doi:

    Blocking of polymerase access to DNA gaps by added integrase. (A) A four-arm substrate was synthesized with two arms matching RSV cDNA sequences (bold lines) and two arms of mixed sequence recapitulating target DNA (thin lines). Two separate molecules with complementary gap sequences were studied to permit assembly of the four-armed structure. Addition of polymerase and a labeled nucleotide (asterisks) results in incorporation of radioactive nucleotides in the gap. Addition of integrase, in contrast, can potentially block access. (B) Polymerization on the RSV gapped dumbbell substrate. Labeled DNA products indicates incorporation of 32 P-labeled dCTPs in the dumbbell substrate by Pol beta. RSV integrase was preincubated for 10 min with the substrate in increasing amounts; lanes 2 to 6 contained 0.1, 1, 10, 50, and 100 ng, respectively. BSA was similarly preincubated with the substrate in lanes 7 to 11 in increasing amounts (0.1, 1, 10, 50, and 100 ng, respectively). The preincubation was followed by the addition of Pol beta and further incubation for 10 min. The amounts of relative incorporation in lanes 2 to 6, respectively, were 100, 108, 34, 15, and 2%. The amounts of relative incorporation in lanes 7 to 11, respectively, were 100, 107, 91, 321, and 108%. (C) Polymerization on a 5′-labeled nicked substrate. The diagrams at the left indicate the unreacted nicked substrate and a strand displacement synthesis product. Reaction compositions were the same as for panel B except for the DNA substrate. The relative percent conversion values for lanes 2 to 6 were, respectively, 100, 113, 115, 87, and 58%. The relative percent conversion values for lanes 7 to 11 were, respectively, 100, 88, 152, 127, and 126%.
    Figure Legend Snippet: Blocking of polymerase access to DNA gaps by added integrase. (A) A four-arm substrate was synthesized with two arms matching RSV cDNA sequences (bold lines) and two arms of mixed sequence recapitulating target DNA (thin lines). Two separate molecules with complementary gap sequences were studied to permit assembly of the four-armed structure. Addition of polymerase and a labeled nucleotide (asterisks) results in incorporation of radioactive nucleotides in the gap. Addition of integrase, in contrast, can potentially block access. (B) Polymerization on the RSV gapped dumbbell substrate. Labeled DNA products indicates incorporation of 32 P-labeled dCTPs in the dumbbell substrate by Pol beta. RSV integrase was preincubated for 10 min with the substrate in increasing amounts; lanes 2 to 6 contained 0.1, 1, 10, 50, and 100 ng, respectively. BSA was similarly preincubated with the substrate in lanes 7 to 11 in increasing amounts (0.1, 1, 10, 50, and 100 ng, respectively). The preincubation was followed by the addition of Pol beta and further incubation for 10 min. The amounts of relative incorporation in lanes 2 to 6, respectively, were 100, 108, 34, 15, and 2%. The amounts of relative incorporation in lanes 7 to 11, respectively, were 100, 107, 91, 321, and 108%. (C) Polymerization on a 5′-labeled nicked substrate. The diagrams at the left indicate the unreacted nicked substrate and a strand displacement synthesis product. Reaction compositions were the same as for panel B except for the DNA substrate. The relative percent conversion values for lanes 2 to 6 were, respectively, 100, 113, 115, 87, and 58%. The relative percent conversion values for lanes 7 to 11 were, respectively, 100, 88, 152, 127, and 126%.

    Techniques Used: Blocking Assay, Synthesized, Sequencing, Labeling, Incubation

    31) 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

    32) Product Images from "Regulation of GC box activity by 8-oxoguanine"

    Article Title: Regulation of GC box activity by 8-oxoguanine

    Journal: Redox Biology

    doi: 10.1016/j.redox.2021.101997

    Incision efficiencies of apurinic lesions in opposite DNA strands of the GC box. (A) Incision of constructs containing dG or the tetrahydrofuran AP lesion (F) at the indicated positions (*) by pure APE1 or the extract of HeLa cells overexpressing the OGG1-GFP fusion protein (the same as analysed in Fig. 3 ). Identical results were obtained with extracts of the parental HeLa cells ( Supplementary Fig. 4 ). (B) Incision by 0.005 units (+) and 1 unit (++) of pure APE1 in the magnesium-containing NEBuffer 4 (upper panel) and the magnesium-free BEH-BSA buffer (lower panel). Constructs with AP lesions (F) were the same as in (A). APE1-resistant constructs containing tetrahydrofuran lesions with phosphorothioate 5′-linkages (SF) were incubated in parallel. (A) and (B) show representative results of three reproducible experiments.
    Figure Legend Snippet: Incision efficiencies of apurinic lesions in opposite DNA strands of the GC box. (A) Incision of constructs containing dG or the tetrahydrofuran AP lesion (F) at the indicated positions (*) by pure APE1 or the extract of HeLa cells overexpressing the OGG1-GFP fusion protein (the same as analysed in Fig. 3 ). Identical results were obtained with extracts of the parental HeLa cells ( Supplementary Fig. 4 ). (B) Incision by 0.005 units (+) and 1 unit (++) of pure APE1 in the magnesium-containing NEBuffer 4 (upper panel) and the magnesium-free BEH-BSA buffer (lower panel). Constructs with AP lesions (F) were the same as in (A). APE1-resistant constructs containing tetrahydrofuran lesions with phosphorothioate 5′-linkages (SF) were incubated in parallel. (A) and (B) show representative results of three reproducible experiments.

    Techniques Used: Construct, Incubation

    33) 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

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    New England Biolabs bsa
    Key elements in the OpenPlant Loop assembly toolkit. (A) The Phytobrick common syntax defines ten <t>DNA</t> part positions and 12 DNA part fusion sites. For Marchantia, we commonly use eight positions and nine fusion sites, by combining positions A2-A3 for proximal promoter (PROMP), and B1-B2 for 5’ untranslated region (5UTR). The other types of parts are: A1 for distal promoter (PROMD), B3 for coding sequence with start codon and no stop codon (CDS1), B4 for coding sequence without start or stop codon (CDS2), B5 coding sequence without start codon and with stop codon (CTAG), B6 for 3’ untranslated region (3UTR) and C1 for transcription terminator (TERM). Parts can span multiple fusion sites, like A1-A3 for promoter (PROM), A1-B2 for promoter with 5’ UTR (PROM5), B3-B4 for coding sequence with start codon and no stop codon for N-terminal fusion with CTAG (CDS12), B3-B6 for coding sequence with start and stop codons (CDS), or B6-C1 for 3’ UTR with terminator (3TERM). (B) Schematic representation of the pUAP4 vector with divergent Sap I sites to accept L0 parts and convergent <t>Bsa</t> I sites to assemble L0 parts into transcription units (L1). (C) Summary of the Loop acceptor vectors of the OpenPlant toolkit. For nuclear genome transformation: pCk (1,2,3,4) can be used for assembly of L0 parts into a Level 1 plasmid using Bsa I, and pCs (A,B,C,E) can be used for assembly of up to four Level 1 plasmids into a Level 2 construct using Sap I. The vectors pCkchlo (1,2,3,4) and pCschlo (A,B,C,E) can be used for chloroplast applications. The vectors L1_lacZgRNA-Ck2, L1_lacZgRNA-Ck3 and L2_lacZgRNA-Cas9 are designed for CRISPR/Cas9 genome editing. LB and RB: left and right border repeats respectively from nopaline C58 T-DNA. Filled blue rounded rectangle: lacZα cassette for blue-white screening. Filled black circles: pSa origin or replication.
    Bsa, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs bsa i hf
    GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent <t>Bsa</t> I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. <t>DNA</t> fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.
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    86
    New England Biolabs bovine serum albumin bsa
    Removal of the inhibitory effects of melanin on <t>PCR</t> amplification. (A) Increasing concentrations of synthetic melanin were added to DNA extracted from 1676 melanoma cell lines. (B) Increasing concentrations of <t>BSA</t> (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.
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    New England Biolabs bovine serum albumin gene bsa
    Antibodies to CAM-cys selectively target purified proteins. A series of purified proteins were treated in the presence or absence of the non-thiol reductant TCEP (2.5mM) with IAM or NEM (5mM). Proteins were then analyzed by Western blotting with 4E7, 52H11, or OX133. Selected CAM-cys labeled proteins reacted with 4E7 and 52H11. Equal loading of proteins was demonstrated by staining membranes with a non-selective protein stain (Revert, LI-COR). Proteins migrated at the expected molecular masses as follows (approximations): beta casein (27 kDa), <t>BSA</t> (65 kDa), BGN (40 kDa), DCN (38 kDa), <t>COL1</t> ( > 250 kDa), COL4 ( > 250 kDa), JAG1 (170 kDa), IL17RC (75 kDa), LAMA2 ( > 180 kDa), vWF (260 kDa), TSP1 (150 kDa), TSP2 (155 kDa).
    Bovine Serum Albumin Gene Bsa, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Key elements in the OpenPlant Loop assembly toolkit. (A) The Phytobrick common syntax defines ten DNA part positions and 12 DNA part fusion sites. For Marchantia, we commonly use eight positions and nine fusion sites, by combining positions A2-A3 for proximal promoter (PROMP), and B1-B2 for 5’ untranslated region (5UTR). The other types of parts are: A1 for distal promoter (PROMD), B3 for coding sequence with start codon and no stop codon (CDS1), B4 for coding sequence without start or stop codon (CDS2), B5 coding sequence without start codon and with stop codon (CTAG), B6 for 3’ untranslated region (3UTR) and C1 for transcription terminator (TERM). Parts can span multiple fusion sites, like A1-A3 for promoter (PROM), A1-B2 for promoter with 5’ UTR (PROM5), B3-B4 for coding sequence with start codon and no stop codon for N-terminal fusion with CTAG (CDS12), B3-B6 for coding sequence with start and stop codons (CDS), or B6-C1 for 3’ UTR with terminator (3TERM). (B) Schematic representation of the pUAP4 vector with divergent Sap I sites to accept L0 parts and convergent Bsa I sites to assemble L0 parts into transcription units (L1). (C) Summary of the Loop acceptor vectors of the OpenPlant toolkit. For nuclear genome transformation: pCk (1,2,3,4) can be used for assembly of L0 parts into a Level 1 plasmid using Bsa I, and pCs (A,B,C,E) can be used for assembly of up to four Level 1 plasmids into a Level 2 construct using Sap I. The vectors pCkchlo (1,2,3,4) and pCschlo (A,B,C,E) can be used for chloroplast applications. The vectors L1_lacZgRNA-Ck2, L1_lacZgRNA-Ck3 and L2_lacZgRNA-Cas9 are designed for CRISPR/Cas9 genome editing. LB and RB: left and right border repeats respectively from nopaline C58 T-DNA. Filled blue rounded rectangle: lacZα cassette for blue-white screening. Filled black circles: pSa origin or replication.

    Journal: bioRxiv

    Article Title: Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha

    doi: 10.1101/2020.02.29.971002

    Figure Lengend Snippet: Key elements in the OpenPlant Loop assembly toolkit. (A) The Phytobrick common syntax defines ten DNA part positions and 12 DNA part fusion sites. For Marchantia, we commonly use eight positions and nine fusion sites, by combining positions A2-A3 for proximal promoter (PROMP), and B1-B2 for 5’ untranslated region (5UTR). The other types of parts are: A1 for distal promoter (PROMD), B3 for coding sequence with start codon and no stop codon (CDS1), B4 for coding sequence without start or stop codon (CDS2), B5 coding sequence without start codon and with stop codon (CTAG), B6 for 3’ untranslated region (3UTR) and C1 for transcription terminator (TERM). Parts can span multiple fusion sites, like A1-A3 for promoter (PROM), A1-B2 for promoter with 5’ UTR (PROM5), B3-B4 for coding sequence with start codon and no stop codon for N-terminal fusion with CTAG (CDS12), B3-B6 for coding sequence with start and stop codons (CDS), or B6-C1 for 3’ UTR with terminator (3TERM). (B) Schematic representation of the pUAP4 vector with divergent Sap I sites to accept L0 parts and convergent Bsa I sites to assemble L0 parts into transcription units (L1). (C) Summary of the Loop acceptor vectors of the OpenPlant toolkit. For nuclear genome transformation: pCk (1,2,3,4) can be used for assembly of L0 parts into a Level 1 plasmid using Bsa I, and pCs (A,B,C,E) can be used for assembly of up to four Level 1 plasmids into a Level 2 construct using Sap I. The vectors pCkchlo (1,2,3,4) and pCschlo (A,B,C,E) can be used for chloroplast applications. The vectors L1_lacZgRNA-Ck2, L1_lacZgRNA-Ck3 and L2_lacZgRNA-Cas9 are designed for CRISPR/Cas9 genome editing. LB and RB: left and right border repeats respectively from nopaline C58 T-DNA. Filled blue rounded rectangle: lacZα cassette for blue-white screening. Filled black circles: pSa origin or replication.

    Article Snippet: Loop assembly Level 1 reaction master mix (MM) contained: 3 µL nuclease-free H2O, 1 µL 10x T4 DNA ligase buffer (New England Biolabs (NEB)), 0.5 µL 1 mg/mL bovine serum albumin (NEB), 0.25 µL T4 DNA ligase at 400 U/µL (NEB), 0.25 µL Bsa I at 10 U/µL (NEB).

    Techniques: Sequencing, Plasmid Preparation, Transformation Assay, Construct, CRISPR

    GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent Bsa I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. DNA fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.

    Journal: PLoS ONE

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    doi: 10.1371/journal.pone.0083043

    Figure Lengend Snippet: GreenGate vector design and layout. A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent Bsa I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two Bsa I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable ccdB gene. DNA fragments can be cloned via the specific overhangs, via the Bam HI and Kpn I sites or via A-overhangs after Xcm I digestion. Plac = lac promoter, SP6 = SP6 promoter, caR = chloramphenicol acetyltransferase gene, T7 = T7 promoter, lacZ = lacZα coding sequence, ampR = beta-lactamase gene, ori = origin of replication. D) Empty destination vector. A counter-selectable ccdB -cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by Bsa I sites, with overhangs A and G. promoter = bacterial promoter. The pSa origin of replication ( ori A. tum. ) requires the presence of the helper plasmid pSOUP in agrobacteria.

    Article Snippet: For the GreenGate reaction itself 1.5 µL plasmid of each of the six modules were mixed with 1 µL of the destination vector, 1.5 µL CutSmart Buffer (alternatively: FastDigest buffer), 1.5 µL ATP (10 mM), 1 µL T4 DNA ligase (30 u/µL) and 1 µL Bsa I-HF (alternatively FastDigest Eco 31I) in a total volume of 15 µL.

    Techniques: Plasmid Preparation, Clone Assay, Transformation Assay, Expressing, Sequencing, Selection, Transgenic Assay

    Multiple expression cassettes on a single T-DNA. A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The Bsa I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show Nicotiana benthamiana leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor ( pUBQ10:B-dummy-ALCR-D-dummy:tRBCS ; pMAS:sulfR:t35S ), the second one ( pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS ) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal Bsa I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the Bsa I recognition sites, since Bsa I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no dcm site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by Bsa I and removal of the Bsa I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) N. benthamiana leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.

    Journal: PLoS ONE

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    doi: 10.1371/journal.pone.0083043

    Figure Lengend Snippet: Multiple expression cassettes on a single T-DNA. A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The Bsa I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show Nicotiana benthamiana leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor ( pUBQ10:B-dummy-ALCR-D-dummy:tRBCS ; pMAS:sulfR:t35S ), the second one ( pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS ) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal Bsa I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the Bsa I recognition sites, since Bsa I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no dcm site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by Bsa I and removal of the Bsa I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) N. benthamiana leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.

    Article Snippet: For the GreenGate reaction itself 1.5 µL plasmid of each of the six modules were mixed with 1 µL of the destination vector, 1.5 µL CutSmart Buffer (alternatively: FastDigest buffer), 1.5 µL ATP (10 mM), 1 µL T4 DNA ligase (30 u/µL) and 1 µL Bsa I-HF (alternatively FastDigest Eco 31I) in a total volume of 15 µL.

    Techniques: Expressing, Plasmid Preparation, Sequencing, Fluorescence, Microscopy, Construct, Methylation, Transformation Assay, Isolation

    The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.

    Journal: PLoS ONE

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    doi: 10.1371/journal.pone.0083043

    Figure Lengend Snippet: The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.

    Article Snippet: For the GreenGate reaction itself 1.5 µL plasmid of each of the six modules were mixed with 1 µL of the destination vector, 1.5 µL CutSmart Buffer (alternatively: FastDigest buffer), 1.5 µL ATP (10 mM), 1 µL T4 DNA ligase (30 u/µL) and 1 µL Bsa I-HF (alternatively FastDigest Eco 31I) in a total volume of 15 µL.

    Techniques: Sequencing, Clone Assay, Plasmid Preparation, Marker, Selection, Generated

    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

    Antibodies to CAM-cys selectively target purified proteins. A series of purified proteins were treated in the presence or absence of the non-thiol reductant TCEP (2.5mM) with IAM or NEM (5mM). Proteins were then analyzed by Western blotting with 4E7, 52H11, or OX133. Selected CAM-cys labeled proteins reacted with 4E7 and 52H11. Equal loading of proteins was demonstrated by staining membranes with a non-selective protein stain (Revert, LI-COR). Proteins migrated at the expected molecular masses as follows (approximations): beta casein (27 kDa), BSA (65 kDa), BGN (40 kDa), DCN (38 kDa), COL1 ( > 250 kDa), COL4 ( > 250 kDa), JAG1 (170 kDa), IL17RC (75 kDa), LAMA2 ( > 180 kDa), vWF (260 kDa), TSP1 (150 kDa), TSP2 (155 kDa).

    Journal: PLoS ONE

    Article Title: Context-dependent monoclonal antibodies against protein carbamidomethyl-cysteine

    doi: 10.1371/journal.pone.0242376

    Figure Lengend Snippet: Antibodies to CAM-cys selectively target purified proteins. A series of purified proteins were treated in the presence or absence of the non-thiol reductant TCEP (2.5mM) with IAM or NEM (5mM). Proteins were then analyzed by Western blotting with 4E7, 52H11, or OX133. Selected CAM-cys labeled proteins reacted with 4E7 and 52H11. Equal loading of proteins was demonstrated by staining membranes with a non-selective protein stain (Revert, LI-COR). Proteins migrated at the expected molecular masses as follows (approximations): beta casein (27 kDa), BSA (65 kDa), BGN (40 kDa), DCN (38 kDa), COL1 ( > 250 kDa), COL4 ( > 250 kDa), JAG1 (170 kDa), IL17RC (75 kDa), LAMA2 ( > 180 kDa), vWF (260 kDa), TSP1 (150 kDa), TSP2 (155 kDa).

    Article Snippet: Purified proteins were purchased from the following sources (all are human except where noted): Beta casein (bovine), Collagen I (gene:COL1), Collagen IV (Gene:COL4) (Sigma-Aldrich); Bovine serum albumin (gene:BSA) (bovine; New England BioLabs); Biglycan (gene:BGN), Decorin (gene:DCN), Jagged 1 (gene:JAG1), Interleukin 17 receptor C (gene:IL17RC), TSP1 and Thrombospondin-2 ((gene:TSP2) (R & D Systems)), Laminin subunit alpha 2 ((gene:LAMA2) (merosin; Millipore), vWF (Haematologic Technologies, Inc).

    Techniques: Chick Chorioallantoic Membrane Assay, Purification, Western Blot, Labeling, Staining