Structured Review

Promega t4 polynucleotide kinase
Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using <t>T4</t> polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.
T4 Polynucleotide Kinase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/t4 polynucleotide kinase/product/Promega
Average 93 stars, based on 52 article reviews
Price from $9.99 to $1999.99
t4 polynucleotide kinase - by Bioz Stars, 2020-07
93/100 stars

Images

1) Product Images from "Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿ †"

Article Title: Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.00011-07

Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using T4 polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.
Figure Legend Snippet: Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using T4 polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.

Techniques Used: In Vivo, Binding Assay, Sequencing, Labeling, End Labeling, Polyacrylamide Gel Electrophoresis

2) Product Images from "Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin"

Article Title: Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin

Journal: EMBO Reports

doi: 10.1038/sj.embor.embor732

Direct labelling of RNA on the 5′ termini of replication intermediates with vaccinia virus guanylyltransferase, an enzyme responsible for messenger RNA capping. ( A ) The 5′ ends of RNA primers attached to P. abyssi replication intermediates were capped with radioactive GTP and samples were fractionated on a sequencing gel (lane 2). Lane 3 contains the same reaction as lane 2, except that total DNA was treated with the restriction endonuclease Eco RI before analysis, confirming that nascent strands were efficiently denatured from template strands. Lane 1, size marker (10-base ladder; Gibco-BRL) labelled with [γ- 32 P]ATP using T4 polynucleotide kinase. ( B ) Schematic diagram of the archaeal Okazaki fragment. The smallest replication intermediate detected (10 nt) is in accordance with the minimum length of RNA primers detected in vitro ).
Figure Legend Snippet: Direct labelling of RNA on the 5′ termini of replication intermediates with vaccinia virus guanylyltransferase, an enzyme responsible for messenger RNA capping. ( A ) The 5′ ends of RNA primers attached to P. abyssi replication intermediates were capped with radioactive GTP and samples were fractionated on a sequencing gel (lane 2). Lane 3 contains the same reaction as lane 2, except that total DNA was treated with the restriction endonuclease Eco RI before analysis, confirming that nascent strands were efficiently denatured from template strands. Lane 1, size marker (10-base ladder; Gibco-BRL) labelled with [γ- 32 P]ATP using T4 polynucleotide kinase. ( B ) Schematic diagram of the archaeal Okazaki fragment. The smallest replication intermediate detected (10 nt) is in accordance with the minimum length of RNA primers detected in vitro ).

Techniques Used: Sequencing, Marker, In Vitro

Unmasking assay showing short replication intermediates in Pyrococcus abyssi and Sulfolobus acidocaldarius . The 5′-OH groups of RNA-primed replication intermediates were selectively exposed by alkaline treatment. 'Unmasked' 5′-OH ends of replication intermediates were labelled with T4 polynucleotide kinase and [γ- 32 P]ATP, and run on an alkaline agarose gel. A control experiment without alkaline treatment is also shown for each experiment. The positions of size markers (100, 200 and 500 nucleotides (nt)) are shown on the left.
Figure Legend Snippet: Unmasking assay showing short replication intermediates in Pyrococcus abyssi and Sulfolobus acidocaldarius . The 5′-OH groups of RNA-primed replication intermediates were selectively exposed by alkaline treatment. 'Unmasked' 5′-OH ends of replication intermediates were labelled with T4 polynucleotide kinase and [γ- 32 P]ATP, and run on an alkaline agarose gel. A control experiment without alkaline treatment is also shown for each experiment. The positions of size markers (100, 200 and 500 nucleotides (nt)) are shown on the left.

Techniques Used: Agarose Gel Electrophoresis

3) Product Images from "RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer"

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer

Journal: Surgery

doi: 10.1016/j.surg.2008.02.005

Determination of protein(s) binding to the 5’-UTR of the OPN promoter region using gel-shift assays (A) Cytoplasmic protein lysates were extracted from HepG2 and Hep3B cells and incubated with (α- 32 P) ATP (2500 Ci/mmol) end-labeled synthesized 5’-UTR from the human OPN promoter sequence using T4 polynucleotide kinase followed by G-50 column purification. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography. Gel is representative of three experiments. (B) Full-length OPN 5’-UTR or deletion in OPN 5’-UTR (58–157) along with OPN promoter region, OPN coding region and 3’-UTR were ligated to the pcDNA3.1 expression vector at the C-terminal followed by transient transfection of HepG2 and Hep3B cells and assayed for OPN protein expression by Western blot analysis. Blot is representative of three experiments.
Figure Legend Snippet: Determination of protein(s) binding to the 5’-UTR of the OPN promoter region using gel-shift assays (A) Cytoplasmic protein lysates were extracted from HepG2 and Hep3B cells and incubated with (α- 32 P) ATP (2500 Ci/mmol) end-labeled synthesized 5’-UTR from the human OPN promoter sequence using T4 polynucleotide kinase followed by G-50 column purification. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography. Gel is representative of three experiments. (B) Full-length OPN 5’-UTR or deletion in OPN 5’-UTR (58–157) along with OPN promoter region, OPN coding region and 3’-UTR were ligated to the pcDNA3.1 expression vector at the C-terminal followed by transient transfection of HepG2 and Hep3B cells and assayed for OPN protein expression by Western blot analysis. Blot is representative of three experiments.

Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Synthesized, Sequencing, Purification, Polyacrylamide Gel Electrophoresis, Autoradiography, Expressing, Plasmid Preparation, Transfection, Western Blot

Related Articles

Agarose Gel Electrophoresis:

Article Title: Sequence-Specific Transcriptional Repression by KS1, a Multiple-Zinc-Finger-Kr?ppel-Associated Box Protein
Article Snippet: .. The PCR product was purified on a 3% low-melting-point agarose gel and end labeled with [γ-32 P]ATP by using T4 polynucleotide kinase according to the manufacturer's suggestions (Promega). .. Gel shift assays were performed using 200 ng of purified GST or GST-KS1 fusion proteins incubated in a buffer containing 20 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2 , 10 μM ZnCl2 , 6% glycerol, 200 μg of bovine serum albumin per ml, and 50 μg of poly(dI-dC)·poly(dI-dC) per ml for 10 min at room temperature.

Synthesized:

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: .. Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification. .. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography.

Labeling:

Article Title: Interaction between CCAAT/Enhancer Binding Protein and Cyclic AMP Response Element Binding Protein 1 Regulates Human Immunodeficiency Virus Type 1 Transcription in Cells of the Monocyte/Macrophage Lineage
Article Snippet: .. Blunt-ended, double-stranded oligonucleotides were end labeled using γ-32 P-labeled ATP and T4 polynucleotide kinase as described by the supplier (Promega). .. The specific activities of the probes used in our electrophoretic mobility shift (EMS) analyses did not generally deviate by a large margin.

Article Title: Hydrostatic and osmotic pressure study of the RNA hydration
Article Snippet: .. 3′-end RNA labeling 6 μL of mixture containing 0.16 mM Cp, 20 μCi [γ-32 P]ATP and 10 U T4 polynucleotide kinase and 1 × PNK buffer (Promega) was incubated at 37°C for 45 min and 99°C for 2 min. Than 0.8 nmol of tRNAPhe and 10 U of T4 RNA ligase (Boehringer) in of 20 μL of ligase buffer (50 mM Tris-HCl pH 8.2, 5 mM ATP, 10% DMSO, 20 μg/mL BSA, 30 mM DTT, 10MgCl2 ,) were added and incubated 16 h at 4°C. .. 5′-end RNA labeling 5 μg of RNA was mix with [γ-32 P]ATP and 6 U T4 polynucleotide kinase (Promega) and kinase buffer (Promega) and incubated 30 min at 37°C.

Article Title: Sequence-Specific Transcriptional Repression by KS1, a Multiple-Zinc-Finger-Kr?ppel-Associated Box Protein
Article Snippet: .. The PCR product was purified on a 3% low-melting-point agarose gel and end labeled with [γ-32 P]ATP by using T4 polynucleotide kinase according to the manufacturer's suggestions (Promega). .. Gel shift assays were performed using 200 ng of purified GST or GST-KS1 fusion proteins incubated in a buffer containing 20 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2 , 10 μM ZnCl2 , 6% glycerol, 200 μg of bovine serum albumin per ml, and 50 μg of poly(dI-dC)·poly(dI-dC) per ml for 10 min at room temperature.

Article Title: Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants in Arabidopsis thaliana
Article Snippet: .. For detection of pre-rRNA precursors and small RNA, 10 pmoles of oligonucleotide primers were 5′end labeled using 50 µCi of [γ32 P] ATP (6000 Ci/mmol) and T4 PNK (Promega). ..

Article Title: Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿ †
Article Snippet: .. The purified DNA fragments (3.5 pmol) were end labeled with 10 U of T4 polynucleotide kinase (Promega) and 10 μCi of [γ32 -P]ATP (Perkin-Elmer, Wellesley, MA) at 37°C for 10 min. .. Various amounts of the purified proteins and probes were incubated in EMSA binding buffer (20 mM KCl, 5% glycerol, 25 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.5 mM dithiothreitol, and 10 ng/ml herring sperm DNA) at 25°C for 10 min in a total volume of 10 μl.

Purification:

Article Title: Sequence-Specific Transcriptional Repression by KS1, a Multiple-Zinc-Finger-Kr?ppel-Associated Box Protein
Article Snippet: .. The PCR product was purified on a 3% low-melting-point agarose gel and end labeled with [γ-32 P]ATP by using T4 polynucleotide kinase according to the manufacturer's suggestions (Promega). .. Gel shift assays were performed using 200 ng of purified GST or GST-KS1 fusion proteins incubated in a buffer containing 20 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2 , 10 μM ZnCl2 , 6% glycerol, 200 μg of bovine serum albumin per ml, and 50 μg of poly(dI-dC)·poly(dI-dC) per ml for 10 min at room temperature.

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: .. Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification. .. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography.

Article Title: Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿ †
Article Snippet: .. The purified DNA fragments (3.5 pmol) were end labeled with 10 U of T4 polynucleotide kinase (Promega) and 10 μCi of [γ32 -P]ATP (Perkin-Elmer, Wellesley, MA) at 37°C for 10 min. .. Various amounts of the purified proteins and probes were incubated in EMSA binding buffer (20 mM KCl, 5% glycerol, 25 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.5 mM dithiothreitol, and 10 ng/ml herring sperm DNA) at 25°C for 10 min in a total volume of 10 μl.

Polymerase Chain Reaction:

Article Title: Sequence-Specific Transcriptional Repression by KS1, a Multiple-Zinc-Finger-Kr?ppel-Associated Box Protein
Article Snippet: .. The PCR product was purified on a 3% low-melting-point agarose gel and end labeled with [γ-32 P]ATP by using T4 polynucleotide kinase according to the manufacturer's suggestions (Promega). .. Gel shift assays were performed using 200 ng of purified GST or GST-KS1 fusion proteins incubated in a buffer containing 20 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2 , 10 μM ZnCl2 , 6% glycerol, 200 μg of bovine serum albumin per ml, and 50 μg of poly(dI-dC)·poly(dI-dC) per ml for 10 min at room temperature.

Incubation:

Article Title: Hydrostatic and osmotic pressure study of the RNA hydration
Article Snippet: .. 3′-end RNA labeling 6 μL of mixture containing 0.16 mM Cp, 20 μCi [γ-32 P]ATP and 10 U T4 polynucleotide kinase and 1 × PNK buffer (Promega) was incubated at 37°C for 45 min and 99°C for 2 min. Than 0.8 nmol of tRNAPhe and 10 U of T4 RNA ligase (Boehringer) in of 20 μL of ligase buffer (50 mM Tris-HCl pH 8.2, 5 mM ATP, 10% DMSO, 20 μg/mL BSA, 30 mM DTT, 10MgCl2 ,) were added and incubated 16 h at 4°C. .. 5′-end RNA labeling 5 μg of RNA was mix with [γ-32 P]ATP and 6 U T4 polynucleotide kinase (Promega) and kinase buffer (Promega) and incubated 30 min at 37°C.

Sequencing:

Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer
Article Snippet: .. Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification. .. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography.

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    Promega t4 polynucleotide kinase
    Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using <t>T4</t> polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.
    T4 Polynucleotide Kinase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 polynucleotide kinase/product/Promega
    Average 93 stars, based on 59 article reviews
    Price from $9.99 to $1999.99
    t4 polynucleotide kinase - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    Image Search Results


    Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using T4 polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.

    Journal: Journal of Bacteriology

    Article Title: Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿Mycobacterial Bacilli Are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling ▿ †

    doi: 10.1128/JB.00011-07

    Figure Lengend Snippet: Transcriptional activities of in vivo-activated genes during chronic tuberculosis. (A) Organization of the rv0348 operon with the secondary structure of the Rv0348 protein. (B) EMSA of the binding ability of rRv0348 or the MBP to an upstream region within the rv0347 sequence. (C) EMSA of the binding ability of rRv0348 in the presence of both specific (rv0347) and nonspecific (map2505) probes. 32 P-labeled probes were prepared by end labeling using T4 polynucleotide kinase. Protein-DNA complexes were resolved in 4% SDS-polyacrylamide gel electrophoresis gels and exposed to X-ray films for 2 to 6 h before development. Probe names and concentrations are listed above each gel image.

    Article Snippet: The purified DNA fragments (3.5 pmol) were end labeled with 10 U of T4 polynucleotide kinase (Promega) and 10 μCi of [γ32 -P]ATP (Perkin-Elmer, Wellesley, MA) at 37°C for 10 min.

    Techniques: In Vivo, Binding Assay, Sequencing, Labeling, End Labeling, Polyacrylamide Gel Electrophoresis

    Direct labelling of RNA on the 5′ termini of replication intermediates with vaccinia virus guanylyltransferase, an enzyme responsible for messenger RNA capping. ( A ) The 5′ ends of RNA primers attached to P. abyssi replication intermediates were capped with radioactive GTP and samples were fractionated on a sequencing gel (lane 2). Lane 3 contains the same reaction as lane 2, except that total DNA was treated with the restriction endonuclease Eco RI before analysis, confirming that nascent strands were efficiently denatured from template strands. Lane 1, size marker (10-base ladder; Gibco-BRL) labelled with [γ- 32 P]ATP using T4 polynucleotide kinase. ( B ) Schematic diagram of the archaeal Okazaki fragment. The smallest replication intermediate detected (10 nt) is in accordance with the minimum length of RNA primers detected in vitro ).

    Journal: EMBO Reports

    Article Title: Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin

    doi: 10.1038/sj.embor.embor732

    Figure Lengend Snippet: Direct labelling of RNA on the 5′ termini of replication intermediates with vaccinia virus guanylyltransferase, an enzyme responsible for messenger RNA capping. ( A ) The 5′ ends of RNA primers attached to P. abyssi replication intermediates were capped with radioactive GTP and samples were fractionated on a sequencing gel (lane 2). Lane 3 contains the same reaction as lane 2, except that total DNA was treated with the restriction endonuclease Eco RI before analysis, confirming that nascent strands were efficiently denatured from template strands. Lane 1, size marker (10-base ladder; Gibco-BRL) labelled with [γ- 32 P]ATP using T4 polynucleotide kinase. ( B ) Schematic diagram of the archaeal Okazaki fragment. The smallest replication intermediate detected (10 nt) is in accordance with the minimum length of RNA primers detected in vitro ).

    Article Snippet: All 5′-OH termini existing in denatured DNA were first 'masked' by treatment for 1 h with 8 units of T4 polynucleotide kinase (PNK; Promega) and non-radioactive ATP at 37 °C in the buffer supplied by the manufacturer.

    Techniques: Sequencing, Marker, In Vitro

    Unmasking assay showing short replication intermediates in Pyrococcus abyssi and Sulfolobus acidocaldarius . The 5′-OH groups of RNA-primed replication intermediates were selectively exposed by alkaline treatment. 'Unmasked' 5′-OH ends of replication intermediates were labelled with T4 polynucleotide kinase and [γ- 32 P]ATP, and run on an alkaline agarose gel. A control experiment without alkaline treatment is also shown for each experiment. The positions of size markers (100, 200 and 500 nucleotides (nt)) are shown on the left.

    Journal: EMBO Reports

    Article Title: Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin

    doi: 10.1038/sj.embor.embor732

    Figure Lengend Snippet: Unmasking assay showing short replication intermediates in Pyrococcus abyssi and Sulfolobus acidocaldarius . The 5′-OH groups of RNA-primed replication intermediates were selectively exposed by alkaline treatment. 'Unmasked' 5′-OH ends of replication intermediates were labelled with T4 polynucleotide kinase and [γ- 32 P]ATP, and run on an alkaline agarose gel. A control experiment without alkaline treatment is also shown for each experiment. The positions of size markers (100, 200 and 500 nucleotides (nt)) are shown on the left.

    Article Snippet: All 5′-OH termini existing in denatured DNA were first 'masked' by treatment for 1 h with 8 units of T4 polynucleotide kinase (PNK; Promega) and non-radioactive ATP at 37 °C in the buffer supplied by the manufacturer.

    Techniques: Agarose Gel Electrophoresis

    Processing of accumulated pre-rRNA in Atnuc-L1 mutant plants is accurate. A) Northern blot analysis using total RNA isolated from WT and Atnuc-L1-1 mutant plants and [γ 32P ] 5′-end labeled primers p34, p35, p36 and p41 to detect 5′ETS1 (lanes 1–3), 5′ETS2 (lanes 4–6), 18S (lanes 7–9) and 3′ETS (lanes 10–12) pre-rRNA sequences respectively. The asterisk and vertical bar indicate expected 5′ETS cleave off and exonucleolityc products (See also Figure S4 ). B) RNAseA/T1 protection analysis was carried out with a radiolabelled probe complementary to the 3′ETS (right). The assay was performed with total RNA from WT (lane 4) and Atnuc-L1 (lanes 5 and 6) or with yeast tRNA as a control (lane 3). A control lane loaded with undigested riboprobe is shown (lane 1). Lane 2, pBR322 digested with HpaII and 5′end labeled with T4 PNK and [γ 32P ] ATP. C) Immunolocalization of fibrillarin in roots from WT and Atnuc-L1-1. Panel mFIB; Fibrillarin appears more abundant in the nucleolus of WT (a) than in the disorganized nucleolus of Atnuc-L1 plants (c) [18] . The nucleolar localization of fibrillarin practically overlaps the localization of AtNUC-L1 (b). Fibrillarin was detected with antibodies against mouse fibrillarin (mFIB 72B9) and Alexa-546 and AtNUC-L1 with antibodies against peptide AtNUC-L1 and Alexa-488. Chromatin in Atnuc-L1-1 is counterstained with DAPI (d). Bar, 10 µm.

    Journal: PLoS Genetics

    Article Title: Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants in Arabidopsis thaliana

    doi: 10.1371/journal.pgen.1001225

    Figure Lengend Snippet: Processing of accumulated pre-rRNA in Atnuc-L1 mutant plants is accurate. A) Northern blot analysis using total RNA isolated from WT and Atnuc-L1-1 mutant plants and [γ 32P ] 5′-end labeled primers p34, p35, p36 and p41 to detect 5′ETS1 (lanes 1–3), 5′ETS2 (lanes 4–6), 18S (lanes 7–9) and 3′ETS (lanes 10–12) pre-rRNA sequences respectively. The asterisk and vertical bar indicate expected 5′ETS cleave off and exonucleolityc products (See also Figure S4 ). B) RNAseA/T1 protection analysis was carried out with a radiolabelled probe complementary to the 3′ETS (right). The assay was performed with total RNA from WT (lane 4) and Atnuc-L1 (lanes 5 and 6) or with yeast tRNA as a control (lane 3). A control lane loaded with undigested riboprobe is shown (lane 1). Lane 2, pBR322 digested with HpaII and 5′end labeled with T4 PNK and [γ 32P ] ATP. C) Immunolocalization of fibrillarin in roots from WT and Atnuc-L1-1. Panel mFIB; Fibrillarin appears more abundant in the nucleolus of WT (a) than in the disorganized nucleolus of Atnuc-L1 plants (c) [18] . The nucleolar localization of fibrillarin practically overlaps the localization of AtNUC-L1 (b). Fibrillarin was detected with antibodies against mouse fibrillarin (mFIB 72B9) and Alexa-546 and AtNUC-L1 with antibodies against peptide AtNUC-L1 and Alexa-488. Chromatin in Atnuc-L1-1 is counterstained with DAPI (d). Bar, 10 µm.

    Article Snippet: For detection of pre-rRNA precursors and small RNA, 10 pmoles of oligonucleotide primers were 5′end labeled using 50 µCi of [γ32 P] ATP (6000 Ci/mmol) and T4 PNK (Promega).

    Techniques: Mutagenesis, Northern Blot, Isolation, Labeling

    Determination of protein(s) binding to the 5’-UTR of the OPN promoter region using gel-shift assays (A) Cytoplasmic protein lysates were extracted from HepG2 and Hep3B cells and incubated with (α- 32 P) ATP (2500 Ci/mmol) end-labeled synthesized 5’-UTR from the human OPN promoter sequence using T4 polynucleotide kinase followed by G-50 column purification. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography. Gel is representative of three experiments. (B) Full-length OPN 5’-UTR or deletion in OPN 5’-UTR (58–157) along with OPN promoter region, OPN coding region and 3’-UTR were ligated to the pcDNA3.1 expression vector at the C-terminal followed by transient transfection of HepG2 and Hep3B cells and assayed for OPN protein expression by Western blot analysis. Blot is representative of three experiments.

    Journal: Surgery

    Article Title: RNA Stability Regulates Differential Expression Of The Metastasis Protein, Osteopontin, In Hepatocellular Cancer

    doi: 10.1016/j.surg.2008.02.005

    Figure Lengend Snippet: Determination of protein(s) binding to the 5’-UTR of the OPN promoter region using gel-shift assays (A) Cytoplasmic protein lysates were extracted from HepG2 and Hep3B cells and incubated with (α- 32 P) ATP (2500 Ci/mmol) end-labeled synthesized 5’-UTR from the human OPN promoter sequence using T4 polynucleotide kinase followed by G-50 column purification. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography. Gel is representative of three experiments. (B) Full-length OPN 5’-UTR or deletion in OPN 5’-UTR (58–157) along with OPN promoter region, OPN coding region and 3’-UTR were ligated to the pcDNA3.1 expression vector at the C-terminal followed by transient transfection of HepG2 and Hep3B cells and assayed for OPN protein expression by Western blot analysis. Blot is representative of three experiments.

    Article Snippet: Briefly, 5’-UTR from the human OPN promoter sequence was synthesized and end-labeled with (α-32 P) ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega Co.) followed by G-50 column purification.

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Synthesized, Sequencing, Purification, Polyacrylamide Gel Electrophoresis, Autoradiography, Expressing, Plasmid Preparation, Transfection, Western Blot