Structured Review

Millipore dnase i
RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), <t>DNase</t> I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.
Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2097 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dnase i/product/Millipore
Average 90 stars, based on 2097 article reviews
Price from $9.99 to $1999.99
dnase i - by Bioz Stars, 2020-04
90/100 stars

Images

1) Product Images from "TRIM25 binds RNA to modulate cellular anti-viral defense"

Article Title: TRIM25 binds RNA to modulate cellular anti-viral defense

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2018.10.003

RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.
Figure Legend Snippet: RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.

Techniques Used: Activity Assay, In Vitro, Purification, Incubation

2) Product Images from "Binding to the Minor Groove of the Double-Strand, Tau Protein Prevents DNA from Damage by Peroxidation"

Article Title: Binding to the Minor Groove of the Double-Strand, Tau Protein Prevents DNA from Damage by Peroxidation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0002600

DNA hydrolysis with DNase I in the presence of tau protein. Tau protein and pEGFP-N1 DNA (quantitative ratio, 1/8) were incubated in 20 mM Tris-HCl buffer containing 2 mM MgCl 2 (pH 8.3, RT, 30 min), and then DNase I (0.05 units) was used to hydrolyze DNA (4730 bp, 100 ng) at 37 °C. Aliquots were taken for agarose gel electrophoresis at different time intervals as indicated. Five mM EDTA (final concentration) was employed to stop the enzymic reaction (lanes 15–20). Hydrolyses of DNA alone (lanes 1–6) and DNA in the presence of BSA (lanes 8–13) were carried out as controls.
Figure Legend Snippet: DNA hydrolysis with DNase I in the presence of tau protein. Tau protein and pEGFP-N1 DNA (quantitative ratio, 1/8) were incubated in 20 mM Tris-HCl buffer containing 2 mM MgCl 2 (pH 8.3, RT, 30 min), and then DNase I (0.05 units) was used to hydrolyze DNA (4730 bp, 100 ng) at 37 °C. Aliquots were taken for agarose gel electrophoresis at different time intervals as indicated. Five mM EDTA (final concentration) was employed to stop the enzymic reaction (lanes 15–20). Hydrolyses of DNA alone (lanes 1–6) and DNA in the presence of BSA (lanes 8–13) were carried out as controls.

Techniques Used: Incubation, Agarose Gel Electrophoresis, Concentration Assay

3) Product Images from "Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence"

Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074662

RFP-LANA associated with GFP-fused histone H1 and H2B. A. Chromosome spreads of 293T cells stably expressing GFP-H1 and GFP-H2B with RFP-LANA. GFP-H1 and GFP-H2B uniformly stained the entire chromosome and RFP-LANA showed distinct punctate localization on the chromosome detected by DAPI staining. B. 293T cells transfected with RFP-LANA and NLS-myc (lane 1), GFP-H1myc (lane 2) and GFP-H2Bmyc (lane 3) were harvested after 48 h post-transfection and lysed in RIPA buffer for immunoprecipitation with anti-myc antibody. Lysates from the above-mentioned transfection were treated with DNase I in second set before anti-myc immunoprecipitation. Bright band of RFP-LANA was detected in Myc-IP panels with GFP-H2Bmyc in untreated (lane 6) as well as DNase I treated panels (lane 12). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the input and IP lanes were detected with anti-myc WB and are indicated with red triangle. C . 10% of the above-transfected cells were passaged and allowed to grow for 96 h before lysing them for anti-myc immunoprecipitation. Lysates were either untreated or DNase I treated before anti-myc immuneprecipitation. Co-precipitating RFP-LANA was detected using anti-LANA western blot (IB:LANA). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the inputs and IP lanes were detected with anti-myc WB (IB:myc).
Figure Legend Snippet: RFP-LANA associated with GFP-fused histone H1 and H2B. A. Chromosome spreads of 293T cells stably expressing GFP-H1 and GFP-H2B with RFP-LANA. GFP-H1 and GFP-H2B uniformly stained the entire chromosome and RFP-LANA showed distinct punctate localization on the chromosome detected by DAPI staining. B. 293T cells transfected with RFP-LANA and NLS-myc (lane 1), GFP-H1myc (lane 2) and GFP-H2Bmyc (lane 3) were harvested after 48 h post-transfection and lysed in RIPA buffer for immunoprecipitation with anti-myc antibody. Lysates from the above-mentioned transfection were treated with DNase I in second set before anti-myc immunoprecipitation. Bright band of RFP-LANA was detected in Myc-IP panels with GFP-H2Bmyc in untreated (lane 6) as well as DNase I treated panels (lane 12). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the input and IP lanes were detected with anti-myc WB and are indicated with red triangle. C . 10% of the above-transfected cells were passaged and allowed to grow for 96 h before lysing them for anti-myc immunoprecipitation. Lysates were either untreated or DNase I treated before anti-myc immuneprecipitation. Co-precipitating RFP-LANA was detected using anti-LANA western blot (IB:LANA). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the inputs and IP lanes were detected with anti-myc WB (IB:myc).

Techniques Used: Stable Transfection, Expressing, Staining, Transfection, Immunoprecipitation, Western Blot

Stably expressing LANA 1–32 aa polypeptide bond to histone H2B. A. Schematic showing the strategy for generating GFP-NLS myc. Oligo containing a Nuclear Localization Sequence (NLS) of EBNA1 with two-tandem myc tag epitope was cloned at BamHI and XbaI sites (MCS) of pEFGCP-C1 vector to generate GFP fused with NLS and myc tag (GFP-NLS-myc). LANA 1–32 aa was PCR amplified with primers flanked with EcoRI and BamHI sites on the 5′ and 3′ respectively. GFP-NLS-myc was digested with EcoRI and BamHI, which released EBNA1 NLS, to clone LANA 1–32 aa (GFP-1–32 aa-myc). B. BJAB stably expressing GFP-NLS-myc or GFP-LANA 1–32myc was subjected for chromosome spreads and the nuclei were stained with propidium iodide (PI). GFP-LANA1–32myc completely painted the chromosomes whereas GFP-NLS-myc localized to nucleus but did not stain the chromosome. C. LANA 1–32 aa sequence showing CBD (5–15 aa) and its alanine substitution mutants highlighted in yellow. D. Histone H1 tagged with HA were transfected with GFP-NLS-myc (control) (lane 1) and GFP-LANA1–32 aa with wt CBD (lane 2) and its alanine substitution mutants (lanes 3–7 corresponding to mutants 3–7 in panel C. WB blot with anti-HA antibody showed a band of H1 with wt CBD LANA indicated with red triangle in the myc IP panel. Input showed uniformed expression of histone H1 in the input lanes. GFP-NLS-myc or GFP-LANA 1–32 and its mutants were detected with anti-myc in the input as well as IP panels. M shows the protein marker lane. Non-specific signals were detected below the red triangle in HA:WB panel. E. HA tagged histone H1 and H2B were co-transfected with GFP-NLS-myc (lane 1) or GFP-LANA1–32 wt (lane 2) and CBD mutants (lanes 3–7 corresponding to the mutants in panel C). Precipitation of GFP-NLS-myc and LANA 1–32 and it mutants showed co-precipitation of H2B (indicated with red triangle) with wt CBD containing LANA 1–32 (lane 2) and relatively lower amount with mutant 14-TG-15 (lane 6). GFP-NLS-myc and GFP-LANA1–32 and its mutants were detected with anti-myc blot in input as well as myc:IP lanes. IgG light chain was detected in HA:WB panel. F. Cells co-transfected with H1-HA and H2B-HA and GFP-NLS-myc or GFP-LANA1–32 aa and its mutants were lysed and the lysates were treated with 50 ug of DNase I for 45 min before immunoprecipitation. Co-precipitating H2B was detected in LANA 1–32 aa with wt CBD (lane 2). Both histones expressed in all the lanes detected by anti-HA WB. GFP-NLS-myc and LANA1–32 along with its mutants were detected with anti-myc WB. IgG light chain was detected in HA:WB panel above the H2B specific band.
Figure Legend Snippet: Stably expressing LANA 1–32 aa polypeptide bond to histone H2B. A. Schematic showing the strategy for generating GFP-NLS myc. Oligo containing a Nuclear Localization Sequence (NLS) of EBNA1 with two-tandem myc tag epitope was cloned at BamHI and XbaI sites (MCS) of pEFGCP-C1 vector to generate GFP fused with NLS and myc tag (GFP-NLS-myc). LANA 1–32 aa was PCR amplified with primers flanked with EcoRI and BamHI sites on the 5′ and 3′ respectively. GFP-NLS-myc was digested with EcoRI and BamHI, which released EBNA1 NLS, to clone LANA 1–32 aa (GFP-1–32 aa-myc). B. BJAB stably expressing GFP-NLS-myc or GFP-LANA 1–32myc was subjected for chromosome spreads and the nuclei were stained with propidium iodide (PI). GFP-LANA1–32myc completely painted the chromosomes whereas GFP-NLS-myc localized to nucleus but did not stain the chromosome. C. LANA 1–32 aa sequence showing CBD (5–15 aa) and its alanine substitution mutants highlighted in yellow. D. Histone H1 tagged with HA were transfected with GFP-NLS-myc (control) (lane 1) and GFP-LANA1–32 aa with wt CBD (lane 2) and its alanine substitution mutants (lanes 3–7 corresponding to mutants 3–7 in panel C. WB blot with anti-HA antibody showed a band of H1 with wt CBD LANA indicated with red triangle in the myc IP panel. Input showed uniformed expression of histone H1 in the input lanes. GFP-NLS-myc or GFP-LANA 1–32 and its mutants were detected with anti-myc in the input as well as IP panels. M shows the protein marker lane. Non-specific signals were detected below the red triangle in HA:WB panel. E. HA tagged histone H1 and H2B were co-transfected with GFP-NLS-myc (lane 1) or GFP-LANA1–32 wt (lane 2) and CBD mutants (lanes 3–7 corresponding to the mutants in panel C). Precipitation of GFP-NLS-myc and LANA 1–32 and it mutants showed co-precipitation of H2B (indicated with red triangle) with wt CBD containing LANA 1–32 (lane 2) and relatively lower amount with mutant 14-TG-15 (lane 6). GFP-NLS-myc and GFP-LANA1–32 and its mutants were detected with anti-myc blot in input as well as myc:IP lanes. IgG light chain was detected in HA:WB panel. F. Cells co-transfected with H1-HA and H2B-HA and GFP-NLS-myc or GFP-LANA1–32 aa and its mutants were lysed and the lysates were treated with 50 ug of DNase I for 45 min before immunoprecipitation. Co-precipitating H2B was detected in LANA 1–32 aa with wt CBD (lane 2). Both histones expressed in all the lanes detected by anti-HA WB. GFP-NLS-myc and LANA1–32 along with its mutants were detected with anti-myc WB. IgG light chain was detected in HA:WB panel above the H2B specific band.

Techniques Used: Stable Transfection, Expressing, Sequencing, Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Staining, Transfection, Western Blot, Marker, Mutagenesis, Immunoprecipitation

Immunoprecipitation of endogenous histones with LANA-N and LANA-FL. A. 293T cells were transfected with GFP-NLS-myc (control) or GFP-LANA-N (1–340 aa)-myc or its alanine substitution mutants (5–7 aa to alanine) and other respective mutants. Lysates were subjected to anti-myc IP without any treatment and immunoprecipitating GFP fusion proteins were detected with anti-myc antibody (Myc:WB panel, red triangle). Endogenous levels of histones were detected using anti-histone H1 (H1:WB) and anti-H2B (H2B:WB) antibodies (indicated red triangle). IgG light chain was detected in H1:WB panel in all the lanes. B. Lysate from 293T cells transfected with GFP-NLS-myc (control) or GFP-LANA-N and its alanine mutants were treated with 45 ug DNase I for 45 min before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected with specific antibodies (red triangle). IgG light chain was detected in H1:WB panel in all the lanes. C. Schematic of LANA-FL with marked CBD (5–15 aa). Mutants 2–6 were alanine substitution mutants of CBD. D. 293 T cells were transfected with myc vector (lane Vec) or myc tagged LANA-FL (lane 1) and its alanine substitution mutants (lanes 2–6). Cell lysate from the transfected cells were subjected for immunoprecipitation with anti-myc antibody followed detection of LANA and its mutants in anti-myc WB (WB:myc). Histone H1 and histone H2B were detected with specific antibodies (red triangle). E. Cells transfected with above plasmid were lysed and the lysates were treated with 45 ug of DNase I before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected using specific antibodies (red triangle).
Figure Legend Snippet: Immunoprecipitation of endogenous histones with LANA-N and LANA-FL. A. 293T cells were transfected with GFP-NLS-myc (control) or GFP-LANA-N (1–340 aa)-myc or its alanine substitution mutants (5–7 aa to alanine) and other respective mutants. Lysates were subjected to anti-myc IP without any treatment and immunoprecipitating GFP fusion proteins were detected with anti-myc antibody (Myc:WB panel, red triangle). Endogenous levels of histones were detected using anti-histone H1 (H1:WB) and anti-H2B (H2B:WB) antibodies (indicated red triangle). IgG light chain was detected in H1:WB panel in all the lanes. B. Lysate from 293T cells transfected with GFP-NLS-myc (control) or GFP-LANA-N and its alanine mutants were treated with 45 ug DNase I for 45 min before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected with specific antibodies (red triangle). IgG light chain was detected in H1:WB panel in all the lanes. C. Schematic of LANA-FL with marked CBD (5–15 aa). Mutants 2–6 were alanine substitution mutants of CBD. D. 293 T cells were transfected with myc vector (lane Vec) or myc tagged LANA-FL (lane 1) and its alanine substitution mutants (lanes 2–6). Cell lysate from the transfected cells were subjected for immunoprecipitation with anti-myc antibody followed detection of LANA and its mutants in anti-myc WB (WB:myc). Histone H1 and histone H2B were detected with specific antibodies (red triangle). E. Cells transfected with above plasmid were lysed and the lysates were treated with 45 ug of DNase I before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected using specific antibodies (red triangle).

Techniques Used: Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation

Analysis of Fluorescence Resonance Energy Transfer (FRET) between LANA and histones. A. 293T cells were stably transduced with CFP-fused H1myc or CFP-fused H2Bmyc. YFP-LANA-Flag was transfected alone in H1-CFP or H2B-CFP expressing cells. To capture the control images for FRET analysis, H1-CFP and H2B-CFP cells were excited with 405 nm laser to detect any bleed through in YFP emission and also signals in FRET channel. Similarly, YFP-LANA was excited with 515 mm to detect the fluorescence in YFP channel as we all FRET Channel. These individually expressing proteins did not show any signals in FRET channel. B. H1-CFP+ LANA-YFP and H2B-CFP+LANA-YFP expressing cells were excited with 405 nm laser to excite CFP proteins, which emits at 477 nm (cyan). Emission spectra of CFP fall in the excitation range of YFP (514 nm). Therefore, based on the proximity of the proteins to transfer energy from the donor to acceptor, emissions from donor can excite the acceptor and thus there is FRET. Emission from CFP fused with H1 and H2B were able to excite YFP-fused with LANA to emit yellow (535 nm) signals. FRET Channel detected almost similar levels of signals in both H1 and H2B transfected with LANA. Co-localized FRET index or FRET efficiency were calculated ImageJ software, which showed comparable localization of H1 and H2B with LANA. C. Proposed model of LANA’s interaction with histone H1 and H2B fused with CFP. For an efficient FRET the intermolecular distance is deciding factor and the molecules separated by less than 10 nm can transfer energy to yield FRET signals. D. Western blot with anti-myc to show the expressions of CFP-fused H1 and H2B and anti-Flag to show comparable expression of YFP-LANA-Flag in those cells. E: Cells used in FRET assay show binding of LANA with histone H1 and H2B after treatment with DNase I as well as MNase. CFP-H1-myc, and CFP-H2B-myc stably expressing in 293T cells were transfected with Flag vector or YFP-LANA-Flag. Lysates from these cells were divided into three parts, i) untreated, ii) MNase treated, iii) DNase I treated followed by immunoprecipitation of LANA with anti-Flag antibody. Immunoprecipitated LANA in all the set was detected with anti-Flag blot (IB:Flag). Co-precipitating H1 and H2B fused to CFP were detected with anti-myc blot (IB:myc). Detection of H1 and H2B in all three conditions (untreated, MNase and DNase I treated) in LANA expressing cells, but not in vector transfected cells, confirmed specific association of both histones with LANA.
Figure Legend Snippet: Analysis of Fluorescence Resonance Energy Transfer (FRET) between LANA and histones. A. 293T cells were stably transduced with CFP-fused H1myc or CFP-fused H2Bmyc. YFP-LANA-Flag was transfected alone in H1-CFP or H2B-CFP expressing cells. To capture the control images for FRET analysis, H1-CFP and H2B-CFP cells were excited with 405 nm laser to detect any bleed through in YFP emission and also signals in FRET channel. Similarly, YFP-LANA was excited with 515 mm to detect the fluorescence in YFP channel as we all FRET Channel. These individually expressing proteins did not show any signals in FRET channel. B. H1-CFP+ LANA-YFP and H2B-CFP+LANA-YFP expressing cells were excited with 405 nm laser to excite CFP proteins, which emits at 477 nm (cyan). Emission spectra of CFP fall in the excitation range of YFP (514 nm). Therefore, based on the proximity of the proteins to transfer energy from the donor to acceptor, emissions from donor can excite the acceptor and thus there is FRET. Emission from CFP fused with H1 and H2B were able to excite YFP-fused with LANA to emit yellow (535 nm) signals. FRET Channel detected almost similar levels of signals in both H1 and H2B transfected with LANA. Co-localized FRET index or FRET efficiency were calculated ImageJ software, which showed comparable localization of H1 and H2B with LANA. C. Proposed model of LANA’s interaction with histone H1 and H2B fused with CFP. For an efficient FRET the intermolecular distance is deciding factor and the molecules separated by less than 10 nm can transfer energy to yield FRET signals. D. Western blot with anti-myc to show the expressions of CFP-fused H1 and H2B and anti-Flag to show comparable expression of YFP-LANA-Flag in those cells. E: Cells used in FRET assay show binding of LANA with histone H1 and H2B after treatment with DNase I as well as MNase. CFP-H1-myc, and CFP-H2B-myc stably expressing in 293T cells were transfected with Flag vector or YFP-LANA-Flag. Lysates from these cells were divided into three parts, i) untreated, ii) MNase treated, iii) DNase I treated followed by immunoprecipitation of LANA with anti-Flag antibody. Immunoprecipitated LANA in all the set was detected with anti-Flag blot (IB:Flag). Co-precipitating H1 and H2B fused to CFP were detected with anti-myc blot (IB:myc). Detection of H1 and H2B in all three conditions (untreated, MNase and DNase I treated) in LANA expressing cells, but not in vector transfected cells, confirmed specific association of both histones with LANA.

Techniques Used: Fluorescence, Förster Resonance Energy Transfer, Stable Transfection, Transduction, Transfection, Expressing, Software, Western Blot, Binding Assay, Plasmid Preparation, Immunoprecipitation

Localization of GFP-fused histone H1 and H2B with LANA in PEL cells. KSHV uninfected (BJAB) and infected (BCBL1 and JSC1) cells were transduced with lentivirus containing GFP-H1myc and GFP-H2Bmyc (schematic shown above panel A). Transduced cells were selected to obtain pure population of cells expressing GFP fused proteins. GFP-NLSmyc was used as control in this assay. A. Chromosome spreads of BCBL1 with GFP-H1 and GFP-H2B shows staining of entire chromosome with green but not in case of control GFP-NLSmyc. B. Chromosome spreads prepared from JSC1 cells stably expressing GFP-H1 and GFP-H2B. C. Chromosome spreads of BJAB cells expressing GFP-H1 and GFP-H2B. LANA were detected in above chromosome spreads using anti-LANA specific antibody. BCBL1 and JSC1 showed punctate dots throughout the chromosome, as expected. Lack of LANA signals in BJAB (KSHV negative cells) confirmed specificity of LANA detection. GFP-H1+LANA and GFP-H2B+LANA panels show yellow spots on the magnified view of the images. D. BCBL1 cells expressing GFP-NLSmyc, GFP-H1myc and GFP-H2Bmyc were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-Myc antibody. Co-precipitating endogenous LANA was detected using anti-LANA antibody (IB:LANA). GFP and myc fused proteins were detected in anti-myc western blot (IB:myc). E. BJAB and BCBL1 cells stably expressing GFP-H1 or GFP-H2B were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-LANA antibody (LANA-IP panels). Co-precipitating GFP-H1 and GFP-H2B were detected with anti-myc antibody (IB:myc).
Figure Legend Snippet: Localization of GFP-fused histone H1 and H2B with LANA in PEL cells. KSHV uninfected (BJAB) and infected (BCBL1 and JSC1) cells were transduced with lentivirus containing GFP-H1myc and GFP-H2Bmyc (schematic shown above panel A). Transduced cells were selected to obtain pure population of cells expressing GFP fused proteins. GFP-NLSmyc was used as control in this assay. A. Chromosome spreads of BCBL1 with GFP-H1 and GFP-H2B shows staining of entire chromosome with green but not in case of control GFP-NLSmyc. B. Chromosome spreads prepared from JSC1 cells stably expressing GFP-H1 and GFP-H2B. C. Chromosome spreads of BJAB cells expressing GFP-H1 and GFP-H2B. LANA were detected in above chromosome spreads using anti-LANA specific antibody. BCBL1 and JSC1 showed punctate dots throughout the chromosome, as expected. Lack of LANA signals in BJAB (KSHV negative cells) confirmed specificity of LANA detection. GFP-H1+LANA and GFP-H2B+LANA panels show yellow spots on the magnified view of the images. D. BCBL1 cells expressing GFP-NLSmyc, GFP-H1myc and GFP-H2Bmyc were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-Myc antibody. Co-precipitating endogenous LANA was detected using anti-LANA antibody (IB:LANA). GFP and myc fused proteins were detected in anti-myc western blot (IB:myc). E. BJAB and BCBL1 cells stably expressing GFP-H1 or GFP-H2B were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-LANA antibody (LANA-IP panels). Co-precipitating GFP-H1 and GFP-H2B were detected with anti-myc antibody (IB:myc).

Techniques Used: Infection, Transduction, Expressing, Staining, Stable Transfection, Immunoprecipitation, Western Blot

Related Articles

Centrifugation:

Article Title: Quorum sensing controls Vibrio cholerae multicellular aggregate formation
Article Snippet: Samples were vigorously shaken for 10 min on a vortex mixer followed by centrifugation for 1 min at 15 000 rpm. .. DNase I (Sigma-Aldrich, catalog # D5025) was added to samples at T = 0 h at a concentration of 100 Kunitz units/mL ( ).

Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection
Article Snippet: The livers were collected and minced into pieces followed by being digested with the buffer containing 1 mg/ml collagenase type IV (Sigma C5138) and 300 U/ml DNAase I (Sigma D5025) at 37 °C for 30 min. Liver homogenates were passed through cell strainer (40 μm), and lysed with RBC lysis buffer to remove the remaining red blood cells. .. The homogenates were further resuspended in 40% (w/v) Percoll-HBSS solution and overlaid onto 70% (w/v) Percoll-HBSS, followed by centrifuging at 2000 rpm for 30 min. After centrifugation, hepatic mononuclear cells (MNC) were collected from the layer of interphase and washed with PBS to remove residual Percoll.

Single-particle Tracking:

Article Title: Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion
Article Snippet: In some rats, signaling antagonists PD98059 (MEK 1/2 and, therefore, ERK 1/2 inhibitor), wortmannin (phosphatidylinositol-3 [PI3] kinase inhibitor), or 8-( p -sulfophenyl)theophylline (SPT) (non-selective adenosine receptor inhibitor) were injected as an intravenous bolus 5 min prior to EndoIII at doses (0.3 mg/kg, 60 μg/kg, and 7.5 mg/kg, respectively) previously determined to block protection from cangrelor [ ]. .. For DNase I studies, we used DNase I type IV (Sigma Aldrich catalogue # D5025) which has specific activity of approximately 2,000 Kunitz units per mg of protein.

Synthesized:

Article Title: Tracking Matrix Effects in the Analysis of DNA Adducts of Polycyclic Aromatic Hydrocarbons
Article Snippet: Reagents including, sodium hydroxide pellets, phosphate buffered saline (PBS) tablets , chloroform, magnesium chloride hexahydrate, Trizma (1 M), deoxyribonucleic acid sodium salt from calf thymus (ctDNA), 2'-deoxyadenosine monohydrate (dA), 2'- deoxycytidine hydrate (dC), thymidine (dT), 2'-deoxyguanosine hydrate (dG), DNase I (bovine pancreas), phosphodiesterase I ( Crotalus adamanteus venom), alkaline phosphatase ( Escherichia coli ), were purchased from Sigma Aldrich (St. Louis, MO). .. The adduct (±)- anti - benzo[ a ]pyrene-7,8-dihydrodiol-9,10-epoxide- N 2 - [15 N5 ]-deoxyguanosine (15 N-BaP-dG) was synthesized in our laboratory [ ].

Blocking Assay:

Article Title: Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion
Article Snippet: In some rats, signaling antagonists PD98059 (MEK 1/2 and, therefore, ERK 1/2 inhibitor), wortmannin (phosphatidylinositol-3 [PI3] kinase inhibitor), or 8-( p -sulfophenyl)theophylline (SPT) (non-selective adenosine receptor inhibitor) were injected as an intravenous bolus 5 min prior to EndoIII at doses (0.3 mg/kg, 60 μg/kg, and 7.5 mg/kg, respectively) previously determined to block protection from cangrelor [ ]. .. For DNase I studies, we used DNase I type IV (Sigma Aldrich catalogue # D5025) which has specific activity of approximately 2,000 Kunitz units per mg of protein.

Incubation:

Article Title: Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle
Article Snippet: A positive control for the TUNEL assay was made by incubating sections for 15 min at 37 °C with 1 μg/mL DNase I (D4263-1VL, SIGMA) in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mg/mL BSA. .. The sections were then blocked for 30 min with 10% normal goat serum (S-1000, Vector) in 2% BSA/PBS and incubated overnight with anti-Ki67 primary mouse monoclonal antibody (clone MIB-1, M7240, Dako) diluted to 0.8 μg/mL.

Article Title: The peptidoglycan and biofilm matrix of Staphylococcus epidermidis undergo structural changes when exposed to human platelets
Article Snippet: Following incubation, supernatants were discarded, and biofilms were washed three times using PBS (pH 7.4). .. Alternatively, to determine the DNA composition of the biofilm matrices, 2.9 ml of 0.5 mg/ml DNase I (DN25; Sigma) in 5 mM MgCl2 was added to grown biofilms for 24h.

Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection
Article Snippet: The livers were collected and minced into pieces followed by being digested with the buffer containing 1 mg/ml collagenase type IV (Sigma C5138) and 300 U/ml DNAase I (Sigma D5025) at 37 °C for 30 min. Liver homogenates were passed through cell strainer (40 μm), and lysed with RBC lysis buffer to remove the remaining red blood cells. .. To determine the IL-17A producing T cells, MNC (5 × 105 /reaction) were incubated with a cocktail of surface marker antibodies including CD3-PE (clone 17A2), CD4-APC (clone GK1.5) and γδ TCR-Brilliant Violet 421(clone GL3).

Activity Assay:

Article Title: Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion
Article Snippet: .. For DNase I studies, we used DNase I type IV (Sigma Aldrich catalogue # D5025) which has specific activity of approximately 2,000 Kunitz units per mg of protein. .. For western blots, antibody to HA tag was obtained from Sigma Aldrich.

Article Title: Bright-field analysis of phi29 DNA packaging motor using a magnetomechanical system
Article Snippet: After visually confirming the existence of the tethered beads, DNase I (Sigma D4527) was introduced to digest the tether DNA. .. Our study shows that the DNA packaging activity of a virus can be visually observed and studied at the single molecular level with a relatively simple system and also visually demonstrates that the phi29 virus can transport a cargo that is four orders of magnitude larger than itself.

Western Blot:

Article Title: Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion
Article Snippet: For DNase I studies, we used DNase I type IV (Sigma Aldrich catalogue # D5025) which has specific activity of approximately 2,000 Kunitz units per mg of protein. .. For western blots, antibody to HA tag was obtained from Sigma Aldrich.

Magnetic Resonance Imaging:

Article Title: Tracking Matrix Effects in the Analysis of DNA Adducts of Polycyclic Aromatic Hydrocarbons
Article Snippet: Reagents including, sodium hydroxide pellets, phosphate buffered saline (PBS) tablets , chloroform, magnesium chloride hexahydrate, Trizma (1 M), deoxyribonucleic acid sodium salt from calf thymus (ctDNA), 2'-deoxyadenosine monohydrate (dA), 2'- deoxycytidine hydrate (dC), thymidine (dT), 2'-deoxyguanosine hydrate (dG), DNase I (bovine pancreas), phosphodiesterase I ( Crotalus adamanteus venom), alkaline phosphatase ( Escherichia coli ), were purchased from Sigma Aldrich (St. Louis, MO). .. The activated carcinogen (±)- anti -benzo[ a ]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) was purchased from MRI Global (Kansas City, MO).

TUNEL Assay:

Article Title: Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle
Article Snippet: .. A positive control for the TUNEL assay was made by incubating sections for 15 min at 37 °C with 1 μg/mL DNase I (D4263-1VL, SIGMA) in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mg/mL BSA. .. The cell proliferation by white adipocytes was detected by immunohistochemical staining for Ki67.

Flow Cytometry:

Article Title: The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin
Article Snippet: .. Treatment of established flow cell biofilms In experiments where EDTA, DNaseI or antibiotic treatment was required, 25 mmol/L EDTA, 100 Kunitz units DNaseI (D5025; Sigma Aldrich Co, St Louis, MO, USA), 50 μ mol/L ampicillin or 1.2 μ mol/L ciprofloxacin were added to 1% CDM in PBS. .. Biofilms were then treated with EDTA, DNaseI or antibiotics for 1 h. After staining with SYTO9®, CLSM images were taken with a Nikon A1 confocal microscope with Ζ-series images taken in 0.5 μ m slices.

Positive Control:

Article Title: Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle
Article Snippet: .. A positive control for the TUNEL assay was made by incubating sections for 15 min at 37 °C with 1 μg/mL DNase I (D4263-1VL, SIGMA) in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mg/mL BSA. .. The cell proliferation by white adipocytes was detected by immunohistochemical staining for Ki67.

Generated:

Article Title: Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion
Article Snippet: An enzymatically inactive K120Q mutant was generated by site-directed mutagenesis using overlap extension [ ]. .. For DNase I studies, we used DNase I type IV (Sigma Aldrich catalogue # D5025) which has specific activity of approximately 2,000 Kunitz units per mg of protein.

other:

Article Title: Mannheimia haemolytica and Its Leukotoxin Cause Neutrophil Extracellular Trap Formation by Bovine Neutrophils ▿
Article Snippet: DNase I (source, bovine pancreas), PMA, E. coli lipopolysaccharide (LPS), and cytochalasin D (Cyto D) were purchased from Sigma-Aldrich (St. Louis, MO).

Injection:

Article Title: Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion
Article Snippet: In some rats, signaling antagonists PD98059 (MEK 1/2 and, therefore, ERK 1/2 inhibitor), wortmannin (phosphatidylinositol-3 [PI3] kinase inhibitor), or 8-( p -sulfophenyl)theophylline (SPT) (non-selective adenosine receptor inhibitor) were injected as an intravenous bolus 5 min prior to EndoIII at doses (0.3 mg/kg, 60 μg/kg, and 7.5 mg/kg, respectively) previously determined to block protection from cangrelor [ ]. .. For DNase I studies, we used DNase I type IV (Sigma Aldrich catalogue # D5025) which has specific activity of approximately 2,000 Kunitz units per mg of protein.

Article Title: Prevention of radiation-induced bystander effects by agents that inactivate cell-free chromatin released from irradiated dying cells
Article Snippet: .. In vivo experiments Bovine pancreatic DNase I (Sigma-Aldrich; Catalogue No- DN25-1G) was injected i.p. at 15 mg/kg twice daily for a duration of 48 h when they were killed (total number of doses received = 5). .. The first dose of DNase I was given 4 hr prior to irradiation.

Article Title: Plasma redox imbalance caused by albumin oxidation promotes lung-predominant NETosis and pulmonary cancer metastasis
Article Snippet: .. For anti-NET therapies, a single intravenous injection of bovine pancreas DNase I (3000 units in 100 μL of saline; Sigma-Aldrich, Canada) was administered on day 2; 6-h prior to injection of cancer cells. .. Cl-amidine (3 mg per mL PBS, 10 mg per kg body weight; MilliporeSigma, MA, USA) was injected intraperitoneally on days 0, 1, and 2.

Staining:

Article Title: Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle
Article Snippet: A positive control for the TUNEL assay was made by incubating sections for 15 min at 37 °C with 1 μg/mL DNase I (D4263-1VL, SIGMA) in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mg/mL BSA. .. The cell proliferation by white adipocytes was detected by immunohistochemical staining for Ki67.

Article Title: The peptidoglycan and biofilm matrix of Staphylococcus epidermidis undergo structural changes when exposed to human platelets
Article Snippet: Alternatively, to determine the DNA composition of the biofilm matrices, 2.9 ml of 0.5 mg/ml DNase I (DN25; Sigma) in 5 mM MgCl2 was added to grown biofilms for 24h. .. Biofilms were rinsed three times with 1X PBS and stained with 0.3% Gram crystal violet dye (BD Biosciences, MD, USA) for 30 min. Staining was eluted with 80:20 ethanol: acetone solution for 15 min. Six 200 μL aliquots of the de-stained solution from each well were transferred to a 96-well polystyrene plate (Falcon, Corning Inc., Durham, NC), and the absorbance at 492 nm was measured with an Expert Plus microplate reader (Biotech, Montreal, Canada).

Article Title: Quorum sensing controls Vibrio cholerae multicellular aggregate formation
Article Snippet: Paragraph title: eDNA quantification and staining ... DNase I (Sigma-Aldrich, catalog # D5025) was added to samples at T = 0 h at a concentration of 100 Kunitz units/mL ( ).

Article Title: The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin
Article Snippet: Treatment of established flow cell biofilms In experiments where EDTA, DNaseI or antibiotic treatment was required, 25 mmol/L EDTA, 100 Kunitz units DNaseI (D5025; Sigma Aldrich Co, St Louis, MO, USA), 50 μ mol/L ampicillin or 1.2 μ mol/L ciprofloxacin were added to 1% CDM in PBS. .. Biofilms were then treated with EDTA, DNaseI or antibiotics for 1 h. After staining with SYTO9®, CLSM images were taken with a Nikon A1 confocal microscope with Ζ-series images taken in 0.5 μ m slices.

Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection
Article Snippet: The livers were collected and minced into pieces followed by being digested with the buffer containing 1 mg/ml collagenase type IV (Sigma C5138) and 300 U/ml DNAase I (Sigma D5025) at 37 °C for 30 min. Liver homogenates were passed through cell strainer (40 μm), and lysed with RBC lysis buffer to remove the remaining red blood cells. .. The isolated hepatic MNC were resuspended in FACS buffer for the following staining.

In Vivo:

Article Title: Prevention of radiation-induced bystander effects by agents that inactivate cell-free chromatin released from irradiated dying cells
Article Snippet: .. In vivo experiments Bovine pancreatic DNase I (Sigma-Aldrich; Catalogue No- DN25-1G) was injected i.p. at 15 mg/kg twice daily for a duration of 48 h when they were killed (total number of doses received = 5). .. The first dose of DNase I was given 4 hr prior to irradiation.

Mutagenesis:

Article Title: Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion
Article Snippet: An enzymatically inactive K120Q mutant was generated by site-directed mutagenesis using overlap extension [ ]. .. For DNase I studies, we used DNase I type IV (Sigma Aldrich catalogue # D5025) which has specific activity of approximately 2,000 Kunitz units per mg of protein.

Isolation:

Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection
Article Snippet: The livers were collected and minced into pieces followed by being digested with the buffer containing 1 mg/ml collagenase type IV (Sigma C5138) and 300 U/ml DNAase I (Sigma D5025) at 37 °C for 30 min. Liver homogenates were passed through cell strainer (40 μm), and lysed with RBC lysis buffer to remove the remaining red blood cells. .. The isolated hepatic MNC were resuspended in FACS buffer for the following staining.

End Labeling:

Article Title: Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle
Article Snippet: Apoptosis and proliferation assays Cell turnover in adipose tissue due to apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. .. A positive control for the TUNEL assay was made by incubating sections for 15 min at 37 °C with 1 μg/mL DNase I (D4263-1VL, SIGMA) in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mg/mL BSA.

Immunohistochemistry:

Article Title: Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle
Article Snippet: A positive control for the TUNEL assay was made by incubating sections for 15 min at 37 °C with 1 μg/mL DNase I (D4263-1VL, SIGMA) in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mg/mL BSA. .. The cell proliferation by white adipocytes was detected by immunohistochemical staining for Ki67.

Microscopy:

Article Title: The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin
Article Snippet: Treatment of established flow cell biofilms In experiments where EDTA, DNaseI or antibiotic treatment was required, 25 mmol/L EDTA, 100 Kunitz units DNaseI (D5025; Sigma Aldrich Co, St Louis, MO, USA), 50 μ mol/L ampicillin or 1.2 μ mol/L ciprofloxacin were added to 1% CDM in PBS. .. Biofilms were then treated with EDTA, DNaseI or antibiotics for 1 h. After staining with SYTO9®, CLSM images were taken with a Nikon A1 confocal microscope with Ζ-series images taken in 0.5 μ m slices.

Mouse Assay:

Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection
Article Snippet: Detection of leukocyte subpopulations in liver by FACS Mice were perfused with PBS buffer containing heparin (10 U/ml) prior tokilling. .. The livers were collected and minced into pieces followed by being digested with the buffer containing 1 mg/ml collagenase type IV (Sigma C5138) and 300 U/ml DNAase I (Sigma D5025) at 37 °C for 30 min. Liver homogenates were passed through cell strainer (40 μm), and lysed with RBC lysis buffer to remove the remaining red blood cells.

Article Title: Plasma redox imbalance caused by albumin oxidation promotes lung-predominant NETosis and pulmonary cancer metastasis
Article Snippet: As shown in Fig. , to induce NETosis, IAA (3 mg per mL saline, 30 mg per kg of body weight) was injected intraperitoneally into 6–8-week-old NSG male mice 2 days prior to the tail vein injection of CAL-33-luciferase (1 × 106 cells in 200 μL PBS). .. For anti-NET therapies, a single intravenous injection of bovine pancreas DNase I (3000 units in 100 μL of saline; Sigma-Aldrich, Canada) was administered on day 2; 6-h prior to injection of cancer cells.

Lysis:

Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection
Article Snippet: .. The livers were collected and minced into pieces followed by being digested with the buffer containing 1 mg/ml collagenase type IV (Sigma C5138) and 300 U/ml DNAase I (Sigma D5025) at 37 °C for 30 min. Liver homogenates were passed through cell strainer (40 μm), and lysed with RBC lysis buffer to remove the remaining red blood cells. .. The homogenates were further resuspended in 40% (w/v) Percoll-HBSS solution and overlaid onto 70% (w/v) Percoll-HBSS, followed by centrifuging at 2000 rpm for 30 min. After centrifugation, hepatic mononuclear cells (MNC) were collected from the layer of interphase and washed with PBS to remove residual Percoll.

Plasmid Preparation:

Article Title: Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle
Article Snippet: A positive control for the TUNEL assay was made by incubating sections for 15 min at 37 °C with 1 μg/mL DNase I (D4263-1VL, SIGMA) in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mg/mL BSA. .. The sections were then blocked for 30 min with 10% normal goat serum (S-1000, Vector) in 2% BSA/PBS and incubated overnight with anti-Ki67 primary mouse monoclonal antibody (clone MIB-1, M7240, Dako) diluted to 0.8 μg/mL.

Software:

Article Title: The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin
Article Snippet: Treatment of established flow cell biofilms In experiments where EDTA, DNaseI or antibiotic treatment was required, 25 mmol/L EDTA, 100 Kunitz units DNaseI (D5025; Sigma Aldrich Co, St Louis, MO, USA), 50 μ mol/L ampicillin or 1.2 μ mol/L ciprofloxacin were added to 1% CDM in PBS. .. Biofilms were volume rendered using IMARIS® software.

In Vitro:

Article Title: Prevention of radiation-induced bystander effects by agents that inactivate cell-free chromatin released from irradiated dying cells
Article Snippet: .. In vitro experiments Bovine pancreatic DNase I (0.005 U; Sigma-Aldrich; Catalogue No- DN25-1G) was used per 1.5 ml of culture media in all experiments. .. In vivo experiments Bovine pancreatic DNase I (Sigma-Aldrich; Catalogue No- DN25-1G) was injected i.p. at 15 mg/kg twice daily for a duration of 48 h when they were killed (total number of doses received = 5).

Ethanol Precipitation:

Article Title: Quorum sensing controls Vibrio cholerae multicellular aggregate formation
Article Snippet: The clarified supernatants were filter sterilized (pore size: 0.22 μm), and DNA was extracted using the standard ethanol precipitation technique ( ; ). .. DNase I (Sigma-Aldrich, catalog # D5025) was added to samples at T = 0 h at a concentration of 100 Kunitz units/mL ( ).

Confocal Laser Scanning Microscopy:

Article Title: The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin
Article Snippet: Treatment of established flow cell biofilms In experiments where EDTA, DNaseI or antibiotic treatment was required, 25 mmol/L EDTA, 100 Kunitz units DNaseI (D5025; Sigma Aldrich Co, St Louis, MO, USA), 50 μ mol/L ampicillin or 1.2 μ mol/L ciprofloxacin were added to 1% CDM in PBS. .. Biofilms were then treated with EDTA, DNaseI or antibiotics for 1 h. After staining with SYTO9®, CLSM images were taken with a Nikon A1 confocal microscope with Ζ-series images taken in 0.5 μ m slices.

Concentration Assay:

Article Title: Prevention of radiation-induced bystander effects by agents that inactivate cell-free chromatin released from irradiated dying cells
Article Snippet: Five milligram tablets of copper were crushed into fine powder and dissolved in distilled water (concentration = 0.04 µg/ml). .. In vivo experiments Bovine pancreatic DNase I (Sigma-Aldrich; Catalogue No- DN25-1G) was injected i.p. at 15 mg/kg twice daily for a duration of 48 h when they were killed (total number of doses received = 5).

Article Title: Quorum sensing controls Vibrio cholerae multicellular aggregate formation
Article Snippet: .. DNase I (Sigma-Aldrich, catalog # D5025) was added to samples at T = 0 h at a concentration of 100 Kunitz units/mL ( ). .. Staining of eDNA was accomplished using the nucleic acid stain TOTO-1 iodide (ThermoFisher, catalog # T3600; final concentration: 1 μM).

Crystal Violet Assay:

Article Title: The peptidoglycan and biofilm matrix of Staphylococcus epidermidis undergo structural changes when exposed to human platelets
Article Snippet: Alternatively, to determine the DNA composition of the biofilm matrices, 2.9 ml of 0.5 mg/ml DNase I (DN25; Sigma) in 5 mM MgCl2 was added to grown biofilms for 24h. .. Following incubation with the respective disruptive agent (proteinase K or DNaseI), residual biofilms were quantified using the semi-quantitative crystal violet assay [ ].

Liquid Chromatography with Mass Spectroscopy:

Article Title: Tracking Matrix Effects in the Analysis of DNA Adducts of Polycyclic Aromatic Hydrocarbons
Article Snippet: Reagents including, sodium hydroxide pellets, phosphate buffered saline (PBS) tablets , chloroform, magnesium chloride hexahydrate, Trizma (1 M), deoxyribonucleic acid sodium salt from calf thymus (ctDNA), 2'-deoxyadenosine monohydrate (dA), 2'- deoxycytidine hydrate (dC), thymidine (dT), 2'-deoxyguanosine hydrate (dG), DNase I (bovine pancreas), phosphodiesterase I ( Crotalus adamanteus venom), alkaline phosphatase ( Escherichia coli ), were purchased from Sigma Aldrich (St. Louis, MO). .. Water, methanol, acetonitrile, ethanol, isopropanol, acetic and formic acids were purchased from Fisher Scientific (Pittsburg, PA) in the highest purity or LC-MS grade when available.

In Situ:

Article Title: Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle
Article Snippet: The paraffin-embedded VAT sections were analyzed with the In situ Apoptosis Detection Kit (MK500, TaKaRa). .. A positive control for the TUNEL assay was made by incubating sections for 15 min at 37 °C with 1 μg/mL DNase I (D4263-1VL, SIGMA) in 50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mg/mL BSA.

Marker:

Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection
Article Snippet: The livers were collected and minced into pieces followed by being digested with the buffer containing 1 mg/ml collagenase type IV (Sigma C5138) and 300 U/ml DNAase I (Sigma D5025) at 37 °C for 30 min. Liver homogenates were passed through cell strainer (40 μm), and lysed with RBC lysis buffer to remove the remaining red blood cells. .. To determine the IL-17A producing T cells, MNC (5 × 105 /reaction) were incubated with a cocktail of surface marker antibodies including CD3-PE (clone 17A2), CD4-APC (clone GK1.5) and γδ TCR-Brilliant Violet 421(clone GL3).

FACS:

Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection
Article Snippet: Paragraph title: Detection of leukocyte subpopulations in liver by FACS ... The livers were collected and minced into pieces followed by being digested with the buffer containing 1 mg/ml collagenase type IV (Sigma C5138) and 300 U/ml DNAase I (Sigma D5025) at 37 °C for 30 min. Liver homogenates were passed through cell strainer (40 μm), and lysed with RBC lysis buffer to remove the remaining red blood cells.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore dnase
    The VP35 IID reduces Th1 responses. A. Schematic representation of the genomes of wt EBOV and mutant EBOVs used to infect cells; mutations R312A in VP35 and K142A in VP24 are indicated by arrows, and the inserted GFP gene is indicated by green rectangles. B. Schematic representation of the cultivation experiment: PBMCs from CMV-positive donors were labeled with CFSE, inoculated with the indicated viruses at MOI of 2 PFU/cell or SEB, and stimulated with CMV pp65 peptides for 4 hours. PBMCs were washed and cultured for 7 days. Thereafter, cells were re-stimulated for 6 hours in culture medium containing CMV pp65 peptides in presence of brefeldin A, monensin, <t>anti-CD28</t> mab, anti-CD49d mab and <t>DNase.</t> As a positive control, cells were stimulated with SEB and treated with phorbol-12-myristate-13 acetate (PMA) and ionomycin. Cells were stained intracellularly for the indicated cytokines followed by multicolor flow cytometry. C. Percentages of total and proliferating (CFSE - ) CD4 + T cells secreting total IFNγ, IL-2 or TNFα normalized to wt EBOV (100%, indicated by the red horizontal lines). Percentages of proliferating and total CD4 + T cells were individually normalized to wt EBOV values in the same donor (100%, indicated by the red horizontal lines). Cells from each individual donor are represented by the same symbol across the panels; mean values are indicated by horizontal bars. Red asterisks indicate significant differences to wt EBOV (p
    Dnase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2768 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase/product/Millipore
    Average 99 stars, based on 2768 article reviews
    Price from $9.99 to $1999.99
    dnase - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    The VP35 IID reduces Th1 responses. A. Schematic representation of the genomes of wt EBOV and mutant EBOVs used to infect cells; mutations R312A in VP35 and K142A in VP24 are indicated by arrows, and the inserted GFP gene is indicated by green rectangles. B. Schematic representation of the cultivation experiment: PBMCs from CMV-positive donors were labeled with CFSE, inoculated with the indicated viruses at MOI of 2 PFU/cell or SEB, and stimulated with CMV pp65 peptides for 4 hours. PBMCs were washed and cultured for 7 days. Thereafter, cells were re-stimulated for 6 hours in culture medium containing CMV pp65 peptides in presence of brefeldin A, monensin, anti-CD28 mab, anti-CD49d mab and DNase. As a positive control, cells were stimulated with SEB and treated with phorbol-12-myristate-13 acetate (PMA) and ionomycin. Cells were stained intracellularly for the indicated cytokines followed by multicolor flow cytometry. C. Percentages of total and proliferating (CFSE - ) CD4 + T cells secreting total IFNγ, IL-2 or TNFα normalized to wt EBOV (100%, indicated by the red horizontal lines). Percentages of proliferating and total CD4 + T cells were individually normalized to wt EBOV values in the same donor (100%, indicated by the red horizontal lines). Cells from each individual donor are represented by the same symbol across the panels; mean values are indicated by horizontal bars. Red asterisks indicate significant differences to wt EBOV (p

    Journal: PLoS Pathogens

    Article Title: The Ebola Interferon Inhibiting Domains Attenuate and Dysregulate Cell-Mediated Immune Responses

    doi: 10.1371/journal.ppat.1006031

    Figure Lengend Snippet: The VP35 IID reduces Th1 responses. A. Schematic representation of the genomes of wt EBOV and mutant EBOVs used to infect cells; mutations R312A in VP35 and K142A in VP24 are indicated by arrows, and the inserted GFP gene is indicated by green rectangles. B. Schematic representation of the cultivation experiment: PBMCs from CMV-positive donors were labeled with CFSE, inoculated with the indicated viruses at MOI of 2 PFU/cell or SEB, and stimulated with CMV pp65 peptides for 4 hours. PBMCs were washed and cultured for 7 days. Thereafter, cells were re-stimulated for 6 hours in culture medium containing CMV pp65 peptides in presence of brefeldin A, monensin, anti-CD28 mab, anti-CD49d mab and DNase. As a positive control, cells were stimulated with SEB and treated with phorbol-12-myristate-13 acetate (PMA) and ionomycin. Cells were stained intracellularly for the indicated cytokines followed by multicolor flow cytometry. C. Percentages of total and proliferating (CFSE - ) CD4 + T cells secreting total IFNγ, IL-2 or TNFα normalized to wt EBOV (100%, indicated by the red horizontal lines). Percentages of proliferating and total CD4 + T cells were individually normalized to wt EBOV values in the same donor (100%, indicated by the red horizontal lines). Cells from each individual donor are represented by the same symbol across the panels; mean values are indicated by horizontal bars. Red asterisks indicate significant differences to wt EBOV (p

    Article Snippet: Stimulation and staining After 7 days of culture, cells were harvested, washed and 2 x 106 cells were stimulated for 6 hours in culture medium with 10 μg/ml Brefeldin A (Sigma-Aldrich), 0.7 μg/ml monensin (GolgiStop, BD Biosciences), 1 μg/ml anti-CD28 (BD Biosciences), 1 μg/ml anti-CD49d (BD Biosciences), 20 μg/ml DNase (Calbiochem) and 2 μg/ml of 15-mer CMV pp65 peptides.

    Techniques: Mutagenesis, Labeling, Cell Culture, Positive Control, Staining, Flow Cytometry, Cytometry

    Nuclear antigenic bridges facilitate histone binding by anti-DNA Abs. Six representative anti-dsDNA/antihistone dual-binding Abs were subjected to DNase-I or sham treatment as detailed in Materials and Methods. Likewise, the histone substrate (i.e., “Antigen”) was also subjected to DNase-I or sham treatment. All Abs were tested for histone reactivity within the same ELISA plates. Horizontal bars represent the mean histone reactivity within each treatment group. The depicted p-values represent the result of comparing each group with the sham-treated (Ab and Ag) control. All DNase-I–treated Abs retained dsDNA-binding after treatment (not depicted).

    Journal: The Journal of Experimental Medicine

    Article Title: Pathogenic Profiles and Molecular Signatures of Antinuclear Autoantibodies Rescued from NZM2410 Lupus Mice

    doi: 10.1084/jem.20030132

    Figure Lengend Snippet: Nuclear antigenic bridges facilitate histone binding by anti-DNA Abs. Six representative anti-dsDNA/antihistone dual-binding Abs were subjected to DNase-I or sham treatment as detailed in Materials and Methods. Likewise, the histone substrate (i.e., “Antigen”) was also subjected to DNase-I or sham treatment. All Abs were tested for histone reactivity within the same ELISA plates. Horizontal bars represent the mean histone reactivity within each treatment group. The depicted p-values represent the result of comparing each group with the sham-treated (Ab and Ag) control. All DNase-I–treated Abs retained dsDNA-binding after treatment (not depicted).

    Article Snippet: Antigen-coated wells were treated with 50 μl of DNase-I (100 μg/ml; Calbiochem) in PBS containing 5 mM MgCl2 and 2 mM CaCl2 at 37°C for 1 h. Sham-treated wells were treated with buffer (PBS) only.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

    Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and DNase I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.

    Journal: Molecular and Cellular Biology

    Article Title: Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF

    doi: 10.1128/MCB.21.6.2221-2234.2001

    Figure Lengend Snippet: Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and DNase I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.

    Article Snippet: Total cell lysates, prepared for loading in sodium dodecyl sulfate (SDS)-containing buffer with brief treatment with DNase I to reduce viscosity as described previously , were run through SDS–10% polyacrylamide gel electrophoresis (PAGE) gels and transferred onto Immobilon P membranes (Millipore, Bedford, Mass.) by semidry blotting.

    Techniques: Methylation, Footprinting

    Role of pal I in MMTV gRNA dimerization, packaging, and propagation. (A) Description of the pal I mutants. The wild type pal I sequence is shown in blue and the mutations are depicted in red. (B) Typical RNA dimerization TBM and TB gels of wild type and mutant MMTV RNAs. M: monomer lane or monomer conformer; D: dimer lane or dimer conformer for each sample. (C) Transfection efficiencies of the mutants and wild type transfer vectors that were used to normalize the packaging efficiency. LUC: Luciferase activity. (D) PCR amplifications of the DNase treated cytoplasmic (panel i) and viral (panel ii) RNAs using virus specific primers. In the third panel (iii), amplification was conducted on the cDNAs obtained from cytoplasmic RNAs using primers that amplify unspliced β-actin mRNA. Multiplex amplifications were conducted in the presence of primers/competimer for 18S ribosomal RNA. The fourth panel (iv) shows PCR of cytoplasmic cDNA using primers that amplify spliced β-actin mRNA. (E) Relative packaging efficiency (RPE) of transfer vector RNAs. (F) Relative hygromycin resistance (Hyg r ) colony forming unit per ml (CFU/ml) for mutant transfer vectors reflecting the relative RNA propagation efficiencies. In (C) , (E) , and (F) , the histograms represent data from at least three independent experiments (± SD). The P values for all mutants in panel (F) were significant (

    Journal: Retrovirology

    Article Title: Structural basis of genomic RNA (gRNA) dimerization and packaging determinants of mouse mammary tumor virus (MMTV)

    doi: 10.1186/s12977-014-0096-6

    Figure Lengend Snippet: Role of pal I in MMTV gRNA dimerization, packaging, and propagation. (A) Description of the pal I mutants. The wild type pal I sequence is shown in blue and the mutations are depicted in red. (B) Typical RNA dimerization TBM and TB gels of wild type and mutant MMTV RNAs. M: monomer lane or monomer conformer; D: dimer lane or dimer conformer for each sample. (C) Transfection efficiencies of the mutants and wild type transfer vectors that were used to normalize the packaging efficiency. LUC: Luciferase activity. (D) PCR amplifications of the DNase treated cytoplasmic (panel i) and viral (panel ii) RNAs using virus specific primers. In the third panel (iii), amplification was conducted on the cDNAs obtained from cytoplasmic RNAs using primers that amplify unspliced β-actin mRNA. Multiplex amplifications were conducted in the presence of primers/competimer for 18S ribosomal RNA. The fourth panel (iv) shows PCR of cytoplasmic cDNA using primers that amplify spliced β-actin mRNA. (E) Relative packaging efficiency (RPE) of transfer vector RNAs. (F) Relative hygromycin resistance (Hyg r ) colony forming unit per ml (CFU/ml) for mutant transfer vectors reflecting the relative RNA propagation efficiencies. In (C) , (E) , and (F) , the histograms represent data from at least three independent experiments (± SD). The P values for all mutants in panel (F) were significant (

    Article Snippet: Following DNase treatment, RNA extraction and purification, the RNAs were concentrated using Amicon Ultra-4 10 K devices (Millipore) and their concentration was determined using nanodrop (ThermoScientific), as described previously [ ].

    Techniques: Sequencing, Mutagenesis, Transfection, Luciferase, Activity Assay, Polymerase Chain Reaction, Amplification, Multiplex Assay, Plasmid Preparation