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Millipore brutp
Colocalization of incorporated BrUMP and MGMT speckles. (a) <t>BrUTP</t> with agarose-encapsulated cells. Pulse-labelling of newly synthesized RNA (or active-transcription sites) by incubating streptolysin <t>O-permeabilized</t> agarose-embedded cells with transcription labelling mixture containing BrUTP. Magnification, ca. ×40. A and B, C and D, and E and F are identical cells stained by MGMT.PAb (in red) and MAb-BrdU (in green; for incorporated BrUMP) simultaneously; note that the cells are spherical in shape due to the embedded agarose. (b) BrUTP with microinjection. Transcription labelling mixture in phosphate-buffered saline was injected into the nuclei of HeLa.CCL2B cells. Injected cells were incubated at 37°C for the required time in minutes before fixation with 4% paraformaldehyde for staining similar to that described for panel a. Magnification, ca. ×90. A and B, C and D, E and F, G and H, I and J, and K and L are identical cells visualized by single-wavelength excitation for MGMT in red and incorporated BrUMP in green, respectively. Pictures K and L are control cells injected with transcription labelling mixture containing UTP instead of BrUTP. (c) Dual-antigen stainings. MGMT and incorporated BrUMP speckles (after 10 min of labelling) were visualized by single (red or green)- and double (“Double exc.” in picture D)-wavelength excitations for half the exposure time of panel c.
Brutp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 16 article reviews
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Images

1) Product Images from "Implication of Localization of Human DNA Repair Enzyme O6-Methylguanine-DNA Methyltransferase at Active Transcription Sites in Transcription-Repair Coupling of the Mutagenic O6-Methylguanine Lesion"

Article Title: Implication of Localization of Human DNA Repair Enzyme O6-Methylguanine-DNA Methyltransferase at Active Transcription Sites in Transcription-Repair Coupling of the Mutagenic O6-Methylguanine Lesion

Journal: Molecular and Cellular Biology

doi:

Colocalization of incorporated BrUMP and MGMT speckles. (a) BrUTP with agarose-encapsulated cells. Pulse-labelling of newly synthesized RNA (or active-transcription sites) by incubating streptolysin O-permeabilized agarose-embedded cells with transcription labelling mixture containing BrUTP. Magnification, ca. ×40. A and B, C and D, and E and F are identical cells stained by MGMT.PAb (in red) and MAb-BrdU (in green; for incorporated BrUMP) simultaneously; note that the cells are spherical in shape due to the embedded agarose. (b) BrUTP with microinjection. Transcription labelling mixture in phosphate-buffered saline was injected into the nuclei of HeLa.CCL2B cells. Injected cells were incubated at 37°C for the required time in minutes before fixation with 4% paraformaldehyde for staining similar to that described for panel a. Magnification, ca. ×90. A and B, C and D, E and F, G and H, I and J, and K and L are identical cells visualized by single-wavelength excitation for MGMT in red and incorporated BrUMP in green, respectively. Pictures K and L are control cells injected with transcription labelling mixture containing UTP instead of BrUTP. (c) Dual-antigen stainings. MGMT and incorporated BrUMP speckles (after 10 min of labelling) were visualized by single (red or green)- and double (“Double exc.” in picture D)-wavelength excitations for half the exposure time of panel c.
Figure Legend Snippet: Colocalization of incorporated BrUMP and MGMT speckles. (a) BrUTP with agarose-encapsulated cells. Pulse-labelling of newly synthesized RNA (or active-transcription sites) by incubating streptolysin O-permeabilized agarose-embedded cells with transcription labelling mixture containing BrUTP. Magnification, ca. ×40. A and B, C and D, and E and F are identical cells stained by MGMT.PAb (in red) and MAb-BrdU (in green; for incorporated BrUMP) simultaneously; note that the cells are spherical in shape due to the embedded agarose. (b) BrUTP with microinjection. Transcription labelling mixture in phosphate-buffered saline was injected into the nuclei of HeLa.CCL2B cells. Injected cells were incubated at 37°C for the required time in minutes before fixation with 4% paraformaldehyde for staining similar to that described for panel a. Magnification, ca. ×90. A and B, C and D, E and F, G and H, I and J, and K and L are identical cells visualized by single-wavelength excitation for MGMT in red and incorporated BrUMP in green, respectively. Pictures K and L are control cells injected with transcription labelling mixture containing UTP instead of BrUTP. (c) Dual-antigen stainings. MGMT and incorporated BrUMP speckles (after 10 min of labelling) were visualized by single (red or green)- and double (“Double exc.” in picture D)-wavelength excitations for half the exposure time of panel c.

Techniques Used: Synthesized, Staining, Injection, Incubation

2) Product Images from "Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells: Evidence that NBs Are Sites of Viral Transcription and Replication ▿"

Article Title: Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells: Evidence that NBs Are Sites of Viral Transcription and Replication ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00554-09

Newly synthesized viral RNAs are present inside the viral IBs. (A) BSR cells were infected at an MOI of 3 for 16 h p.i., transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein (as a marker of the Negri bodies) was detected by using the rabbit polyclonal anti-P antibody, followed by incubation with Alexa 568-goat anti-rabbit IgG (P). Newly synthesized RNAs were detected by using mouse MAb anti-BrdU antibody (BrUTP) and goat anti-mouse Alexa 488. Colocalization of the viral RNA (vRNA) with the IBs is shown in the merged panel in the presence of the drug (+ActD). In cells untreated with ActD, cellular mRNA is produced in vast excess over vRNA. Thus, for these images, the PMT gain of the confocal laser scanning microscope is lower, so the signal for vRNA is lower than for that shown in the presence of the drug, and specific colocalization is not readily observed. The scale bars correspond to 12 μm. (B) BrUTP signals are specific to RNA produced by rabies virus. BSR cells were infected with the VTF7-3 recombinant virus and cotransfected with plasmids expressing N and P proteins. Cells were then transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein and RNA were detected as in panel A. Note that in the absence of the drug, no BrUTP signal is detectable in the IBs formed by N and P proteins, but some RNA signals are detected in the cytoplasmic inclusions, most probably due to vaccinia virus infection (-ActD). In the presence of the drug, no RNA labeling is observed (+ActD).
Figure Legend Snippet: Newly synthesized viral RNAs are present inside the viral IBs. (A) BSR cells were infected at an MOI of 3 for 16 h p.i., transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein (as a marker of the Negri bodies) was detected by using the rabbit polyclonal anti-P antibody, followed by incubation with Alexa 568-goat anti-rabbit IgG (P). Newly synthesized RNAs were detected by using mouse MAb anti-BrdU antibody (BrUTP) and goat anti-mouse Alexa 488. Colocalization of the viral RNA (vRNA) with the IBs is shown in the merged panel in the presence of the drug (+ActD). In cells untreated with ActD, cellular mRNA is produced in vast excess over vRNA. Thus, for these images, the PMT gain of the confocal laser scanning microscope is lower, so the signal for vRNA is lower than for that shown in the presence of the drug, and specific colocalization is not readily observed. The scale bars correspond to 12 μm. (B) BrUTP signals are specific to RNA produced by rabies virus. BSR cells were infected with the VTF7-3 recombinant virus and cotransfected with plasmids expressing N and P proteins. Cells were then transfected with BrUTP for 20 min in the presence (+ActD) or absence (-ActD) of ActD. P protein and RNA were detected as in panel A. Note that in the absence of the drug, no BrUTP signal is detectable in the IBs formed by N and P proteins, but some RNA signals are detected in the cytoplasmic inclusions, most probably due to vaccinia virus infection (-ActD). In the presence of the drug, no RNA labeling is observed (+ActD).

Techniques Used: Synthesized, Infection, Transfection, Marker, Incubation, Produced, Laser-Scanning Microscopy, Recombinant, Expressing, Labeling

3) Product Images from "Blocking Variant Surface Glycoprotein Synthesis in Trypanosoma brucei Triggers a General Arrest in Translation Initiation"

Article Title: Blocking Variant Surface Glycoprotein Synthesis in Trypanosoma brucei Triggers a General Arrest in Translation Initiation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0007532

Transcription analysis of cells where VSG synthesis is blocked. A) Northern blot analysis of T. brucei 221VG1.1 cells where VSG221 RNAi had been induced for the time indicated above in hours (h). The parental (P) T. brucei 90-13 cell line does not contain the VSG221 RNAi construct. The T. brucei 221VG1.1 cell line was either not incubated with tetracycline (− Tet) or had VSG221 RNAi induced with tetracycline for the times indicated above. Blots were hybridised with probes for Actin, NUP1 , eGFP (present in the active VSG221 ES), TDP1 , tubulin (Tub), ESAG5 , 18S rRNA, ISWI , ESAG6/7, VSG221 and PFR2 . Ethidium stains of the gels are indicated on the right to indicate total RNA loaded. B) Transcription analysis of T. brucei 221VG1.1 where VSG synthesis was blocked by the induction of VSG221 RNAi with tetracycline (Tet) for 0 or 24 hours (h). Cells were incubated with BrUTP to label nascent transcripts and subsequently incubated with an anti-BrdUTP antibody, and a secondary antibody coupled to Alexa 488. DNA was stained with DAPI. A normal precytokinesis cell (0 h) is compared with a precytokinesis cell arising after the induction of VSG RNAi for 24 hours (24 h). The experiment was performed in the absence of α-amanitin (− α-ama) to visualise total transcription, or in the presence of 200 µg ml −1 α-amanitin to inhibit transcription by RNA polymerases II and III and visualise transcription by RNA polymerase I (+ α-ama). The scale bar indicates 4 µm. Quantitation of transcription as fluorescence in the FITC channel is in arbitrary units using 50 cells per time point, with standard deviation indicated with error bars.
Figure Legend Snippet: Transcription analysis of cells where VSG synthesis is blocked. A) Northern blot analysis of T. brucei 221VG1.1 cells where VSG221 RNAi had been induced for the time indicated above in hours (h). The parental (P) T. brucei 90-13 cell line does not contain the VSG221 RNAi construct. The T. brucei 221VG1.1 cell line was either not incubated with tetracycline (− Tet) or had VSG221 RNAi induced with tetracycline for the times indicated above. Blots were hybridised with probes for Actin, NUP1 , eGFP (present in the active VSG221 ES), TDP1 , tubulin (Tub), ESAG5 , 18S rRNA, ISWI , ESAG6/7, VSG221 and PFR2 . Ethidium stains of the gels are indicated on the right to indicate total RNA loaded. B) Transcription analysis of T. brucei 221VG1.1 where VSG synthesis was blocked by the induction of VSG221 RNAi with tetracycline (Tet) for 0 or 24 hours (h). Cells were incubated with BrUTP to label nascent transcripts and subsequently incubated with an anti-BrdUTP antibody, and a secondary antibody coupled to Alexa 488. DNA was stained with DAPI. A normal precytokinesis cell (0 h) is compared with a precytokinesis cell arising after the induction of VSG RNAi for 24 hours (24 h). The experiment was performed in the absence of α-amanitin (− α-ama) to visualise total transcription, or in the presence of 200 µg ml −1 α-amanitin to inhibit transcription by RNA polymerases II and III and visualise transcription by RNA polymerase I (+ α-ama). The scale bar indicates 4 µm. Quantitation of transcription as fluorescence in the FITC channel is in arbitrary units using 50 cells per time point, with standard deviation indicated with error bars.

Techniques Used: Northern Blot, Construct, Incubation, Staining, Quantitation Assay, Fluorescence, Standard Deviation

Related Articles

Transfection:

Article Title: Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells: Evidence that NBs Are Sites of Viral Transcription and Replication ▿
Article Snippet: .. Briefly, the infected cells (16 h p.i.) were treated with 15 μg of ActD/ml for 1 h and then transfected with BrUTP (B 7166; Sigma) at a final concentration of 10 mM by using Lipofectamine 2000 (Invitrogen) and maintained in the presence of ActD for 20 min. .. P protein was detected by using the rabbit polyclonal anti-P antibody, followed by incubation with Alexa 568-conjugated goat anti-rabbit IgG.

Article Title: Microtubule-dependent Organization of Vaccinia Virus Core-derivd Early mRNAs into Distinct Cytoplasmic Structures
Article Snippet: .. Briefly, 10 mM of 5′-bromouridine 5′-triphosphate (BrUTP; Sigma) or 2 μg of an antisense oligonucleotide corresponding to the H5R gene (2′- O -methylated, 5′-GCCAUCUUUGUGAAACUAGUAUC-3′) coupled to four biotin moieties was preincubated for 30 min in 30 μl of transfection medium containing 3.7 μl of lipofectin. .. The samples were diluted in 300 μl of transfection medium and added to the cells in presence of 5 μg/ml act D. After 1-h incubation, the cells were extensively washed and incubated in complete culture medium containing 5 mM hydroxyurea (Sigma) and fixed at the indicated times post-act D washout.

In Vitro:

Article Title: Long noncoding RNA DRAIC inhibits prostate cancer progression by interacting with IKK to inhibit NF-κB activation
Article Snippet: .. The sense and antisense DRAIC RNA was labeled by random incorporation of 5-Bromo-UTP during the in vitro transcription. .. 5 μg of each sense and antisense in vitro transcribed DRAIC RNA was used per pull down experiment.

Immunoprecipitation:

Article Title: Hierarchical Molecular Events Driven by Oocyte-Specific Factors Lead to Rapid and Extensive Reprogramming
Article Snippet: .. RNA Immunoprecipitation BrUTP (Sigma: B7166, 4.6 nl of 100 mM stock) was injected to the cytoplasm of Xenopus oocytes 2 hr after transplantation with MEFs. .. Oocytes were collected 48 hr after NT, and RNA was extracted using a QIAGEN RNeasy kit (eight oocytes per column).

Methylation:

Article Title: Microtubule-dependent Organization of Vaccinia Virus Core-derivd Early mRNAs into Distinct Cytoplasmic Structures
Article Snippet: .. Briefly, 10 mM of 5′-bromouridine 5′-triphosphate (BrUTP; Sigma) or 2 μg of an antisense oligonucleotide corresponding to the H5R gene (2′- O -methylated, 5′-GCCAUCUUUGUGAAACUAGUAUC-3′) coupled to four biotin moieties was preincubated for 30 min in 30 μl of transfection medium containing 3.7 μl of lipofectin. .. The samples were diluted in 300 μl of transfection medium and added to the cells in presence of 5 μg/ml act D. After 1-h incubation, the cells were extensively washed and incubated in complete culture medium containing 5 mM hydroxyurea (Sigma) and fixed at the indicated times post-act D washout.

Infection:

Article Title: Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells: Evidence that NBs Are Sites of Viral Transcription and Replication ▿
Article Snippet: .. Briefly, the infected cells (16 h p.i.) were treated with 15 μg of ActD/ml for 1 h and then transfected with BrUTP (B 7166; Sigma) at a final concentration of 10 mM by using Lipofectamine 2000 (Invitrogen) and maintained in the presence of ActD for 20 min. .. P protein was detected by using the rabbit polyclonal anti-P antibody, followed by incubation with Alexa 568-conjugated goat anti-rabbit IgG.

Concentration Assay:

Article Title: Functional Characterization of Negri Bodies (NBs) in Rabies Virus-Infected Cells: Evidence that NBs Are Sites of Viral Transcription and Replication ▿
Article Snippet: .. Briefly, the infected cells (16 h p.i.) were treated with 15 μg of ActD/ml for 1 h and then transfected with BrUTP (B 7166; Sigma) at a final concentration of 10 mM by using Lipofectamine 2000 (Invitrogen) and maintained in the presence of ActD for 20 min. .. P protein was detected by using the rabbit polyclonal anti-P antibody, followed by incubation with Alexa 568-conjugated goat anti-rabbit IgG.

Article Title: Nonphosphorylated Human La Antigen Interacts with Nucleolin at Nucleolar Sites Involved in rRNA Biogenesis
Article Snippet: .. Cells were washed, permeabilized, and incubated for 10 min at 37°C in transcription buffer containing 0.5 mM 5-bromouridine 5′-triphosphate (BrUTP; Sigma) containing the appropriate concentration of α-amanitin and processed as described previously with primary monoclonal Ab against BrUTP (1:200; Caltag) and AbNP (1:50) in phosphate-buffered saline for 1 h, washed with phosphate-buffered saline, and incubated with secondary Ab conjugated with fluorescein or Texas red (Vector Laboratories) ( , ). .. Fluorescence resonance energy transfer (FRET) experiments were performed in transfected HeLa cells on a Zeiss 510 confocal microscope with a ×100 and 1.3 numerical aperture planapochromat oil objective and ×3 zoom.

Incubation:

Article Title: Chromosome intermingling—the physical basis of chromosome organization in differentiated cells
Article Snippet: .. This was followed by incubation with 10 mM Br-UTP (Sigma, B7166) in hypotonic buffer at room temperature. .. After 5 min, the hypotonic media was replaced with 10% FBS containing DMEM medium and incubated at 37°C and 5% CO2 for 20 min before fixing it with freshly thawed 4% PFA (in PBS) for 10 min.

Article Title: Nonphosphorylated Human La Antigen Interacts with Nucleolin at Nucleolar Sites Involved in rRNA Biogenesis
Article Snippet: .. Cells were washed, permeabilized, and incubated for 10 min at 37°C in transcription buffer containing 0.5 mM 5-bromouridine 5′-triphosphate (BrUTP; Sigma) containing the appropriate concentration of α-amanitin and processed as described previously with primary monoclonal Ab against BrUTP (1:200; Caltag) and AbNP (1:50) in phosphate-buffered saline for 1 h, washed with phosphate-buffered saline, and incubated with secondary Ab conjugated with fluorescein or Texas red (Vector Laboratories) ( , ). .. Fluorescence resonance energy transfer (FRET) experiments were performed in transfected HeLa cells on a Zeiss 510 confocal microscope with a ×100 and 1.3 numerical aperture planapochromat oil objective and ×3 zoom.

Labeling:

Article Title: Long noncoding RNA DRAIC inhibits prostate cancer progression by interacting with IKK to inhibit NF-κB activation
Article Snippet: .. The sense and antisense DRAIC RNA was labeled by random incorporation of 5-Bromo-UTP during the in vitro transcription. .. 5 μg of each sense and antisense in vitro transcribed DRAIC RNA was used per pull down experiment.

Transplantation Assay:

Article Title: Hierarchical Molecular Events Driven by Oocyte-Specific Factors Lead to Rapid and Extensive Reprogramming
Article Snippet: .. RNA Immunoprecipitation BrUTP (Sigma: B7166, 4.6 nl of 100 mM stock) was injected to the cytoplasm of Xenopus oocytes 2 hr after transplantation with MEFs. .. Oocytes were collected 48 hr after NT, and RNA was extracted using a QIAGEN RNeasy kit (eight oocytes per column).

Injection:

Article Title: Hierarchical Molecular Events Driven by Oocyte-Specific Factors Lead to Rapid and Extensive Reprogramming
Article Snippet: .. RNA Immunoprecipitation BrUTP (Sigma: B7166, 4.6 nl of 100 mM stock) was injected to the cytoplasm of Xenopus oocytes 2 hr after transplantation with MEFs. .. Oocytes were collected 48 hr after NT, and RNA was extracted using a QIAGEN RNeasy kit (eight oocytes per column).

Article Title: Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways
Article Snippet: .. Using a Drummond injector (Drummond Nanoject, Drummond Scientific Company, USA), the required amount of mRNAs or gRNAs (9nl at 1mg/ml), or BrUTP (Sigma: B7166, 4.6nl at 100mM), is injected in the cytoplasm from the vegetal pole of the recipient oocyte. .. Injected oocytes are cultured in 1X MBS, P/S, 0.2%BSA at 18°C.

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    Millipore brutp
    Brutp, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/brutp/product/Millipore
    Average 90 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    brutp - by Bioz Stars, 2020-09
    90/100 stars
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