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FUJIFILM ethylenediaminetetraacetic acid
Ethylenediaminetetraacetic Acid, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ethylenediaminetetraacetic acid - by Bioz Stars, 2020-03
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Related Articles

Diagnostic Assay:

Article Title: Fetuin-Mineral Complex Reflects Extraosseous Calcification Stress in CKD
Article Snippet: Portions of pellets were dissolved in 15 μl of 150 mM HCl for calcium analysis ( C) using clinical diagnostic reagents (Wako, Osaka, Japan). .. Other portions of pellets were dissolved in 80 μl of Laemmli buffer containing 60 mM EDTA, and 15-μl portions were resolved by 10 to 20% Super-Sep polyacrylamide gel (Wako, Osaka, Japan) electrophoresis and then stained with Coomassie Brilliant Blue.

Centrifugation:

Article Title: Fetuin-Mineral Complex Reflects Extraosseous Calcification Stress in CKD
Article Snippet: The supernatant was discarded, and the tubes were rinsed twice with 1 ml of 150 mM NaCl and separated again by centrifugation for 5 minutes at 16,000 × g . .. Other portions of pellets were dissolved in 80 μl of Laemmli buffer containing 60 mM EDTA, and 15-μl portions were resolved by 10 to 20% Super-Sep polyacrylamide gel (Wako, Osaka, Japan) electrophoresis and then stained with Coomassie Brilliant Blue.

Positive Control:

Article Title: Diarylethenes Display In Vitro Anti-TB Activity and Are Efficient Hits Targeting the Mycobacterium tuberculosis HU Protein
Article Snippet: The compounds 1 – 4 were added in increasing concentrations and further incubated for 5 min. SD4 was used at 5 µM as a positive control. .. All reactions were analysed in 6% (w /v ) non-denaturing polyacrylamide gels (29.4:0.6 acrylamide:bisacrylamide) in TBE buffer [45 mM Tris−borate (pH 8.3) and 1 mM EDTA] and visualised using a phosphorimager (Fujifilm, Tokyo, Japan).

Autoradiography:

Article Title: NAD+-dependent synthesis of a 5′-phospho-ADP-ribosylated RNA/DNA cap by RNA 2′-phosphotransferase Tpt1
Article Snippet: .. The products were analyzed by electrophoresis (at 55 W constant power) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA and visualized by autoradiography and/or scanning the gel with a Fujifilm FLA-7000 imaging device. ..

Article Title: Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain
Article Snippet: .. The products were analyzed by electrophoresis (at a constant power of 55 W) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA, visualized by autoradiography, and quantified by scanning the gel with a Fujifilm FLA-7000 imager. ..

Cytometry:

Article Title: A Cancer-specific Monoclonal Antibody Recognizes the Aberrantly Glycosylated Podoplanin
Article Snippet: .. Flow cytometry Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Wako Pure Chemical Industries Ltd.) . .. After washing with phosphate buffered saline (PBS), the cells were treated with primary antibodies (1 μg/ml) for 30 min at 4°C, followed by treatment with Oregon green-conjugated anti-mouse IgG (Life Technologies Corp.).

Enzyme-linked Immunosorbent Assay:

Article Title: Fetuin-Mineral Complex Reflects Extraosseous Calcification Stress in CKD
Article Snippet: Serum samples (50 μl) were centrifuged for 2 hours at 4°C at 16,000 × g , and then fetuin-A levels in the supernatant fraction were measured using the Human Fetuin-A ELISA kits. .. Other portions of pellets were dissolved in 80 μl of Laemmli buffer containing 60 mM EDTA, and 15-μl portions were resolved by 10 to 20% Super-Sep polyacrylamide gel (Wako, Osaka, Japan) electrophoresis and then stained with Coomassie Brilliant Blue.

Incubation:

Article Title: NAD+-dependent synthesis of a 5′-phospho-ADP-ribosylated RNA/DNA cap by RNA 2′-phosphotransferase Tpt1
Article Snippet: Assay of Tpt1 activity Reaction mixtures containing 100 mM Tris–HCl (pH 7.5), 5′ 32 P-labeled oligonucleotide substrates, NAD+ , and Tpt1 as specified in the figure legends were incubated at 37°C. .. The products were analyzed by electrophoresis (at 55 W constant power) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA and visualized by autoradiography and/or scanning the gel with a Fujifilm FLA-7000 imaging device.

Article Title: Diarylethenes Display In Vitro Anti-TB Activity and Are Efficient Hits Targeting the Mycobacterium tuberculosis HU Protein
Article Snippet: The compounds 1 – 4 were added in increasing concentrations and further incubated for 5 min. SD4 was used at 5 µM as a positive control. .. All reactions were analysed in 6% (w /v ) non-denaturing polyacrylamide gels (29.4:0.6 acrylamide:bisacrylamide) in TBE buffer [45 mM Tris−borate (pH 8.3) and 1 mM EDTA] and visualised using a phosphorimager (Fujifilm, Tokyo, Japan).

Article Title: Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain
Article Snippet: Reactions mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl2 or NiCl2 , 100 nM (1 pmol) 10-mer 5′-32 P-labeled pDNAp substrate (5′-pATCACGCTTCp; prepared by enzymatic phosphorylation of a synthetic 10-mer HO DNAp oligonucleotide by phosphatase-inactive T4 Pnkp-D167N and then gel purification), and HD-Pnk as specified were incubated at 37°C. .. The products were analyzed by electrophoresis (at a constant power of 55 W) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA, visualized by autoradiography, and quantified by scanning the gel with a Fujifilm FLA-7000 imager.

Article Title: Structure-Function Analysis of the Phosphoesterase Component of the Nucleic Acid End-Healing Enzyme Runella slithyformis HD-Pnk
Article Snippet: Reactions mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl2 , 100 nM (1 pmol) 10-mer 5′ 32 P-labeled pDNAp substrate (5′-pATCACGCTTCp; prepared by enzymatic phosphorylation of a synthetic 10-mer HO DNAp oligonucleotide by phosphatase-dead T4 Pnkp-D167N and then gel purified), and HD-Pnk as specified were incubated at 37°C for 10 min. .. The products were analyzed by electrophoresis at 55 W constant power through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate–1 mM EDTA, visualized by scanning the gel with a FujiFilm FLA-7000 imager, and quantified using ImageJ software.

Article Title: Reversible inhibition of the blood-testis barrier protein improves stem cell homing in mouse testes
Article Snippet: .. For colchicine (20 μM), cytochalasin D (100 μM, both from Calbiochem), EDTA (20 mM, Wako), or CPE (0.5 mg/ml; a gift from Dr Sachiko Tsukita, Osaka University, Osaka, Japan) treatment, donor cells were incubated with the indicated reagent and microinjected into the seminiferous tubules. .. For Cldn5 overexpression experiments, approximately 10 μl of lentivirus particles, produced by transient expression of CSII-EF- Cldn5 -IRES2-Puro (3 × 108 infectious units/ml), were microinjected into the seminiferous tubules of busulfan-treated B6 seminiferous tubules.

Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
Article Snippet: .. For DNA gel shift assays, 20 nM of 5′-end P32 -labeled 20-nt double-stranded DNA (5′-ACTGGACAAATACTCCGAGG-3′) were incubated with different concentrations of purified recombinant protein in a buffer containing 20 mM HEPES (pH 7.0) and 5 mM EDTA on ice for 1 h After incubation, the reaction mixtures were resolved in 10% polyacrylamide gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system ( , ). .. For the DNase activity assays shown in , and , 309-bp linear double-stranded DNA (30 nM) were incubated with the purified protein (1 μM) in the DNase reaction buffer containing 20 mM HEPES (pH 7.0), 100 mM NaCl, 1 mM CaCl2 , and 1 mM DTT at 37°C for the indicated periods.

Activity Assay:

Article Title: NAD+-dependent synthesis of a 5′-phospho-ADP-ribosylated RNA/DNA cap by RNA 2′-phosphotransferase Tpt1
Article Snippet: Paragraph title: Assay of Tpt1 activity ... The products were analyzed by electrophoresis (at 55 W constant power) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA and visualized by autoradiography and/or scanning the gel with a Fujifilm FLA-7000 imaging device.

Article Title: Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain
Article Snippet: Paragraph title: Assay of DNA 3′ phosphatase activity. ... The products were analyzed by electrophoresis (at a constant power of 55 W) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA, visualized by autoradiography, and quantified by scanning the gel with a Fujifilm FLA-7000 imager.

Article Title: Structure-Function Analysis of the Phosphoesterase Component of the Nucleic Acid End-Healing Enzyme Runella slithyformis HD-Pnk
Article Snippet: Paragraph title: Assay of DNA 3′ phosphatase activity. ... The products were analyzed by electrophoresis at 55 W constant power through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate–1 mM EDTA, visualized by scanning the gel with a FujiFilm FLA-7000 imager, and quantified using ImageJ software.

Article Title: Leukocyte-derived extracellular DNA contributes to abnormal pressure elevation in the extracorporeal circulation circuit
Article Snippet: .. For inhibiting endogenous DNase activity, EDTA (Wako) was added to the priming solution at a concentration of 5 mM. .. Monitoring of in-circuit pressure In each experiment, 100 mL of porcine blood was administered into the reservoir and the flow rate of blood–saline solution was kept at 1 L/min.

Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
Article Snippet: Paragraph title: DNA-binding and DNase activity assays ... For DNA gel shift assays, 20 nM of 5′-end P32 -labeled 20-nt double-stranded DNA (5′-ACTGGACAAATACTCCGAGG-3′) were incubated with different concentrations of purified recombinant protein in a buffer containing 20 mM HEPES (pH 7.0) and 5 mM EDTA on ice for 1 h After incubation, the reaction mixtures were resolved in 10% polyacrylamide gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system ( , ).

Expressing:

Article Title: Reversible inhibition of the blood-testis barrier protein improves stem cell homing in mouse testes
Article Snippet: For colchicine (20 μM), cytochalasin D (100 μM, both from Calbiochem), EDTA (20 mM, Wako), or CPE (0.5 mg/ml; a gift from Dr Sachiko Tsukita, Osaka University, Osaka, Japan) treatment, donor cells were incubated with the indicated reagent and microinjected into the seminiferous tubules. .. For Cldn5 overexpression experiments, approximately 10 μl of lentivirus particles, produced by transient expression of CSII-EF- Cldn5 -IRES2-Puro (3 × 108 infectious units/ml), were microinjected into the seminiferous tubules of busulfan-treated B6 seminiferous tubules.

Transplantation Assay:

Article Title: Reversible inhibition of the blood-testis barrier protein improves stem cell homing in mouse testes
Article Snippet: Paragraph title: Animals and spermatogonial transplantation ... For colchicine (20 μM), cytochalasin D (100 μM, both from Calbiochem), EDTA (20 mM, Wako), or CPE (0.5 mg/ml; a gift from Dr Sachiko Tsukita, Osaka University, Osaka, Japan) treatment, donor cells were incubated with the indicated reagent and microinjected into the seminiferous tubules.

Over Expression:

Article Title: Reversible inhibition of the blood-testis barrier protein improves stem cell homing in mouse testes
Article Snippet: For colchicine (20 μM), cytochalasin D (100 μM, both from Calbiochem), EDTA (20 mM, Wako), or CPE (0.5 mg/ml; a gift from Dr Sachiko Tsukita, Osaka University, Osaka, Japan) treatment, donor cells were incubated with the indicated reagent and microinjected into the seminiferous tubules. .. For Cldn5 overexpression experiments, approximately 10 μl of lentivirus particles, produced by transient expression of CSII-EF- Cldn5 -IRES2-Puro (3 × 108 infectious units/ml), were microinjected into the seminiferous tubules of busulfan-treated B6 seminiferous tubules.

Gel Purification:

Article Title: Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain
Article Snippet: Reactions mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl2 or NiCl2 , 100 nM (1 pmol) 10-mer 5′-32 P-labeled pDNAp substrate (5′-pATCACGCTTCp; prepared by enzymatic phosphorylation of a synthetic 10-mer HO DNAp oligonucleotide by phosphatase-inactive T4 Pnkp-D167N and then gel purification), and HD-Pnk as specified were incubated at 37°C. .. The products were analyzed by electrophoresis (at a constant power of 55 W) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA, visualized by autoradiography, and quantified by scanning the gel with a Fujifilm FLA-7000 imager.

Flow Cytometry:

Article Title: A Cancer-specific Monoclonal Antibody Recognizes the Aberrantly Glycosylated Podoplanin
Article Snippet: .. Flow cytometry Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Wako Pure Chemical Industries Ltd.) . .. After washing with phosphate buffered saline (PBS), the cells were treated with primary antibodies (1 μg/ml) for 30 min at 4°C, followed by treatment with Oregon green-conjugated anti-mouse IgG (Life Technologies Corp.).

Cell Culture:

Article Title: Development of selective cytotoxic viral vectors for concentration of undifferentiated cells in cardiomyocytes derived from human induced pluripotent stem cells
Article Snippet: .. Colonies were passaged by dissociating the single cells once every 3–4 days using 0.5 mM EDTA in PBS at a density of 2 × 104 cells/cm2 . imCM spiked with hiPSCs were cultured on laminin-521 in mTeSR1 medium. hiPSC-derived cardiomyocytes (Cellular Dynamics International; CDI, Madison, WI, USA) were cultured on 0.1% gelatin (StemCell) or laminin-521-coated plates in iCell Cardiomyocytes Maintenance Medium (CDI). .. Viral infection Cells were infected with AdV and AAV vectors at 10 infection units (IUs) per cell and 1 × 105 viral genome copies per cells, respectively.

Article Title: Spontaneous formation of neutrophil extracellular traps in serum‐free culture conditions
Article Snippet: .. When indicated, neutrophils were stimulated with 20 or 200 nm PMA, or cultured in the presence of 100 U·mL−1 DNase I (TaKaRa, Osaka, Japan), 40 mg·mL−1 bovine serum albumin (BSA; Wako Pure Chemical Industries, Osaka, Japan), 40 mg·mL−1 human serum albumin (Wako), heat‐treated (complement‐inactivated) serum, protein G/A sepharose‐treated (immunoglobulin‐depleted) serum, 10 mm EDTA, or 5 or 20 mm N ‐acetyl‐l ‐cysteine (NAC; Wako). .. Bacteria‐trapping assay Neutrophils (1 × 106 cells·mL−1 ) were cultured for 1 h in a serum‐free condition to induce the formation of NETs.

Inhibition:

Article Title: Diarylethenes Display In Vitro Anti-TB Activity and Are Efficient Hits Targeting the Mycobacterium tuberculosis HU Protein
Article Snippet: Paragraph title: 4.5. Mtb-HU Inhibition ... All reactions were analysed in 6% (w /v ) non-denaturing polyacrylamide gels (29.4:0.6 acrylamide:bisacrylamide) in TBE buffer [45 mM Tris−borate (pH 8.3) and 1 mM EDTA] and visualised using a phosphorimager (Fujifilm, Tokyo, Japan).

Imaging:

Article Title: NAD+-dependent synthesis of a 5′-phospho-ADP-ribosylated RNA/DNA cap by RNA 2′-phosphotransferase Tpt1
Article Snippet: .. The products were analyzed by electrophoresis (at 55 W constant power) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA and visualized by autoradiography and/or scanning the gel with a Fujifilm FLA-7000 imaging device. ..

Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
Article Snippet: .. For DNA gel shift assays, 20 nM of 5′-end P32 -labeled 20-nt double-stranded DNA (5′-ACTGGACAAATACTCCGAGG-3′) were incubated with different concentrations of purified recombinant protein in a buffer containing 20 mM HEPES (pH 7.0) and 5 mM EDTA on ice for 1 h After incubation, the reaction mixtures were resolved in 10% polyacrylamide gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system ( , ). .. For the DNase activity assays shown in , and , 309-bp linear double-stranded DNA (30 nM) were incubated with the purified protein (1 μM) in the DNase reaction buffer containing 20 mM HEPES (pH 7.0), 100 mM NaCl, 1 mM CaCl2 , and 1 mM DTT at 37°C for the indicated periods.

Injection:

Article Title: Reversible inhibition of the blood-testis barrier protein improves stem cell homing in mouse testes
Article Snippet: Each injection filled 75−85% of the seminiferous tubules. .. For colchicine (20 μM), cytochalasin D (100 μM, both from Calbiochem), EDTA (20 mM, Wako), or CPE (0.5 mg/ml; a gift from Dr Sachiko Tsukita, Osaka University, Osaka, Japan) treatment, donor cells were incubated with the indicated reagent and microinjected into the seminiferous tubules.

Recombinant:

Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
Article Snippet: .. For DNA gel shift assays, 20 nM of 5′-end P32 -labeled 20-nt double-stranded DNA (5′-ACTGGACAAATACTCCGAGG-3′) were incubated with different concentrations of purified recombinant protein in a buffer containing 20 mM HEPES (pH 7.0) and 5 mM EDTA on ice for 1 h After incubation, the reaction mixtures were resolved in 10% polyacrylamide gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system ( , ). .. For the DNase activity assays shown in , and , 309-bp linear double-stranded DNA (30 nM) were incubated with the purified protein (1 μM) in the DNase reaction buffer containing 20 mM HEPES (pH 7.0), 100 mM NaCl, 1 mM CaCl2 , and 1 mM DTT at 37°C for the indicated periods.

Fluorescence:

Article Title: A Cancer-specific Monoclonal Antibody Recognizes the Aberrantly Glycosylated Podoplanin
Article Snippet: Flow cytometry Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Wako Pure Chemical Industries Ltd.) . .. Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

Article Title: Spontaneous formation of neutrophil extracellular traps in serum‐free culture conditions
Article Snippet: Fluorescence was quantified using a microplate reader equipped with filters to detect excitation/emission maxima of 504/523 nm (EnSpire; PerkinElmer, Waltham, MA, USA). .. When indicated, neutrophils were stimulated with 20 or 200 nm PMA, or cultured in the presence of 100 U·mL−1 DNase I (TaKaRa, Osaka, Japan), 40 mg·mL−1 bovine serum albumin (BSA; Wako Pure Chemical Industries, Osaka, Japan), 40 mg·mL−1 human serum albumin (Wako), heat‐treated (complement‐inactivated) serum, protein G/A sepharose‐treated (immunoglobulin‐depleted) serum, 10 mm EDTA, or 5 or 20 mm N ‐acetyl‐l ‐cysteine (NAC; Wako).

Labeling:

Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
Article Snippet: .. For DNA gel shift assays, 20 nM of 5′-end P32 -labeled 20-nt double-stranded DNA (5′-ACTGGACAAATACTCCGAGG-3′) were incubated with different concentrations of purified recombinant protein in a buffer containing 20 mM HEPES (pH 7.0) and 5 mM EDTA on ice for 1 h After incubation, the reaction mixtures were resolved in 10% polyacrylamide gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system ( , ). .. For the DNase activity assays shown in , and , 309-bp linear double-stranded DNA (30 nM) were incubated with the purified protein (1 μM) in the DNase reaction buffer containing 20 mM HEPES (pH 7.0), 100 mM NaCl, 1 mM CaCl2 , and 1 mM DTT at 37°C for the indicated periods.

Mouse Assay:

Article Title: Reversible inhibition of the blood-testis barrier protein improves stem cell homing in mouse testes
Article Snippet: Approximately 4 or 10 μl of cell suspension was microinjected into the testes of W and busulfan-treated mice, respectively. .. For colchicine (20 μM), cytochalasin D (100 μM, both from Calbiochem), EDTA (20 mM, Wako), or CPE (0.5 mg/ml; a gift from Dr Sachiko Tsukita, Osaka University, Osaka, Japan) treatment, donor cells were incubated with the indicated reagent and microinjected into the seminiferous tubules.

Electrophoretic Mobility Shift Assay:

Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
Article Snippet: .. For DNA gel shift assays, 20 nM of 5′-end P32 -labeled 20-nt double-stranded DNA (5′-ACTGGACAAATACTCCGAGG-3′) were incubated with different concentrations of purified recombinant protein in a buffer containing 20 mM HEPES (pH 7.0) and 5 mM EDTA on ice for 1 h After incubation, the reaction mixtures were resolved in 10% polyacrylamide gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system ( , ). .. For the DNase activity assays shown in , and , 309-bp linear double-stranded DNA (30 nM) were incubated with the purified protein (1 μM) in the DNase reaction buffer containing 20 mM HEPES (pH 7.0), 100 mM NaCl, 1 mM CaCl2 , and 1 mM DTT at 37°C for the indicated periods.

Purification:

Article Title: Structure-Function Analysis of the Phosphoesterase Component of the Nucleic Acid End-Healing Enzyme Runella slithyformis HD-Pnk
Article Snippet: Reactions mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl2 , 100 nM (1 pmol) 10-mer 5′ 32 P-labeled pDNAp substrate (5′-pATCACGCTTCp; prepared by enzymatic phosphorylation of a synthetic 10-mer HO DNAp oligonucleotide by phosphatase-dead T4 Pnkp-D167N and then gel purified), and HD-Pnk as specified were incubated at 37°C for 10 min. .. The products were analyzed by electrophoresis at 55 W constant power through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate–1 mM EDTA, visualized by scanning the gel with a FujiFilm FLA-7000 imager, and quantified using ImageJ software.

Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
Article Snippet: .. For DNA gel shift assays, 20 nM of 5′-end P32 -labeled 20-nt double-stranded DNA (5′-ACTGGACAAATACTCCGAGG-3′) were incubated with different concentrations of purified recombinant protein in a buffer containing 20 mM HEPES (pH 7.0) and 5 mM EDTA on ice for 1 h After incubation, the reaction mixtures were resolved in 10% polyacrylamide gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system ( , ). .. For the DNase activity assays shown in , and , 309-bp linear double-stranded DNA (30 nM) were incubated with the purified protein (1 μM) in the DNase reaction buffer containing 20 mM HEPES (pH 7.0), 100 mM NaCl, 1 mM CaCl2 , and 1 mM DTT at 37°C for the indicated periods.

Plasmid Preparation:

Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
Article Snippet: For DNA gel shift assays, 20 nM of 5′-end P32 -labeled 20-nt double-stranded DNA (5′-ACTGGACAAATACTCCGAGG-3′) were incubated with different concentrations of purified recombinant protein in a buffer containing 20 mM HEPES (pH 7.0) and 5 mM EDTA on ice for 1 h After incubation, the reaction mixtures were resolved in 10% polyacrylamide gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system ( , ). .. For the DNase activity assays shown in , a pQE-70 plasmid DNA (100 ng) was incubated with 0.2 μM purified protein in the DNase reaction buffer at 37°C for the indicated periods.

Software:

Article Title: Structure-Function Analysis of the Phosphoesterase Component of the Nucleic Acid End-Healing Enzyme Runella slithyformis HD-Pnk
Article Snippet: .. The products were analyzed by electrophoresis at 55 W constant power through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate–1 mM EDTA, visualized by scanning the gel with a FujiFilm FLA-7000 imager, and quantified using ImageJ software. ..

Electrophoresis:

Article Title: NAD+-dependent synthesis of a 5′-phospho-ADP-ribosylated RNA/DNA cap by RNA 2′-phosphotransferase Tpt1
Article Snippet: .. The products were analyzed by electrophoresis (at 55 W constant power) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA and visualized by autoradiography and/or scanning the gel with a Fujifilm FLA-7000 imaging device. ..

Article Title: Fetuin-Mineral Complex Reflects Extraosseous Calcification Stress in CKD
Article Snippet: .. Other portions of pellets were dissolved in 80 μl of Laemmli buffer containing 60 mM EDTA, and 15-μl portions were resolved by 10 to 20% Super-Sep polyacrylamide gel (Wako, Osaka, Japan) electrophoresis and then stained with Coomassie Brilliant Blue. .. Gel pieces containing proteins of interest were excised and subjected to in-gel digestion with sequencing-grade modified trypsin (Promega, Madison, WI).

Article Title: Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain
Article Snippet: .. The products were analyzed by electrophoresis (at a constant power of 55 W) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA, visualized by autoradiography, and quantified by scanning the gel with a Fujifilm FLA-7000 imager. ..

Article Title: Structure-Function Analysis of the Phosphoesterase Component of the Nucleic Acid End-Healing Enzyme Runella slithyformis HD-Pnk
Article Snippet: .. The products were analyzed by electrophoresis at 55 W constant power through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate–1 mM EDTA, visualized by scanning the gel with a FujiFilm FLA-7000 imager, and quantified using ImageJ software. ..

Produced:

Article Title: Reversible inhibition of the blood-testis barrier protein improves stem cell homing in mouse testes
Article Snippet: For colchicine (20 μM), cytochalasin D (100 μM, both from Calbiochem), EDTA (20 mM, Wako), or CPE (0.5 mg/ml; a gift from Dr Sachiko Tsukita, Osaka University, Osaka, Japan) treatment, donor cells were incubated with the indicated reagent and microinjected into the seminiferous tubules. .. For Cldn5 overexpression experiments, approximately 10 μl of lentivirus particles, produced by transient expression of CSII-EF- Cldn5 -IRES2-Puro (3 × 108 infectious units/ml), were microinjected into the seminiferous tubules of busulfan-treated B6 seminiferous tubules.

Concentration Assay:

Article Title: Leukocyte-derived extracellular DNA contributes to abnormal pressure elevation in the extracorporeal circulation circuit
Article Snippet: .. For inhibiting endogenous DNase activity, EDTA (Wako) was added to the priming solution at a concentration of 5 mM. .. Monitoring of in-circuit pressure In each experiment, 100 mL of porcine blood was administered into the reservoir and the flow rate of blood–saline solution was kept at 1 L/min.

Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
Article Snippet: For DNA gel shift assays, 20 nM of 5′-end P32 -labeled 20-nt double-stranded DNA (5′-ACTGGACAAATACTCCGAGG-3′) were incubated with different concentrations of purified recombinant protein in a buffer containing 20 mM HEPES (pH 7.0) and 5 mM EDTA on ice for 1 h After incubation, the reaction mixtures were resolved in 10% polyacrylamide gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system ( , ). .. For the DNase activity assays shown in , purified CRN-4 and/or CRN-5 (concentration 1–2 μM) was incubated with 309-bp double-stranded DNA (30 nM) in a solution of 10 mM NaCl, 3 mM MgCl2 , 1 mM CaCl2 , and 20 mM Tris-HCl (pH 6.0) at 30°C for 40 min. All the reactions were stopped by adding 10 mM proteinase K for 10 min to remove proteins.

Staining:

Article Title: Fetuin-Mineral Complex Reflects Extraosseous Calcification Stress in CKD
Article Snippet: .. Other portions of pellets were dissolved in 80 μl of Laemmli buffer containing 60 mM EDTA, and 15-μl portions were resolved by 10 to 20% Super-Sep polyacrylamide gel (Wako, Osaka, Japan) electrophoresis and then stained with Coomassie Brilliant Blue. .. Gel pieces containing proteins of interest were excised and subjected to in-gel digestion with sequencing-grade modified trypsin (Promega, Madison, WI).

Article Title: Spontaneous formation of neutrophil extracellular traps in serum‐free culture conditions
Article Snippet: Quantification of NETs Extracellular DNA was stained with 5 μm SYTOX Green (Life Technologies, Carlsbad, CA, USA), a fluorescent membrane‐impermeable DNA dye. .. When indicated, neutrophils were stimulated with 20 or 200 nm PMA, or cultured in the presence of 100 U·mL−1 DNase I (TaKaRa, Osaka, Japan), 40 mg·mL−1 bovine serum albumin (BSA; Wako Pure Chemical Industries, Osaka, Japan), 40 mg·mL−1 human serum albumin (Wako), heat‐treated (complement‐inactivated) serum, protein G/A sepharose‐treated (immunoglobulin‐depleted) serum, 10 mm EDTA, or 5 or 20 mm N ‐acetyl‐l ‐cysteine (NAC; Wako).

other:

Article Title: Effect of Melatonin on Human Dental Papilla Cells
Article Snippet: Extirpated tooth germs were fixed with 10 N Mildform® (Wako Pure Chemical Industries, Ltd., Osaka, Japan), decalcified in 10% EDTA (Wako), and embedded in paraffin.

Article Title: High Content Screening in hESC-Neural Progenitors Identifies Drug Candidates that Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain
Article Snippet: To induce forebrain organoid differentiation, H9 hESC line (WA-09, WiCell) hPSCs were dissociated to single cells using EDTA, and 9000 cells were reaggregated using low-adhesion V-bottom 96 well plates (Wako) in Essential8 Medium (Fisher Scientific) with 10 μM Y-27632 (Tocris Biosciences).

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    FUJIFILM edta
    HD-Pnk is a copper-dependent p -nitrophenylphosphatase. (A) Metal specificity. Reaction mixtures (25 μl) containing 100 mM <t>Tris-acetate</t> (pH 6.0), 10 mM (250 nmol) p -nitrophenylphosphate, 10 pmol (0.4 μM) of HD-Pnk, and either no added metal cofactor (lane –) or 1 mM the indicated divalent cation (added as chloride salt) were incubated at 37°C for 10 min. The extents of p -nitrophenol product formation are plotted in bar graph format. Each datum is the average from three separate experiments ± the standard errors of the mean (SEM). (B) Metal concentration dependence. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 10 mM (250 nmol) p -nitrophenylphosphate, 20 pmol (0.8 μM) of HD-Pnk, and 0, 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, or 5 mM CuCl 2 or NiCl 2 as indicated were incubated at 37°C for 10 min. p -Nitrophenol formation is plotted as a function of divalent cation concentration. (C) His73 is essential for p -nitrophenylphosphatase activity. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 10 mM (250 nmol) p -nitrophenylphosphate, and 10 pmol (0.4 μM) wild-type (WT) HD-Pnk or mutants H73A or D254A as specified were incubated at 37°C for 10 min. (D) Enzyme titration. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 10 mM (250 nmol) p -nitrophenylphosphate, and 0, 1. 2.5, 5, 7.5, 10, or 20 pmol of HD-Pnk were incubated at 37°C for 10 min. p -Nitrophenol formation is plotted as a function of input enzyme. Each datum is the average from three separate experiments ± the SEM. (E) Time course. A reaction mixture (250 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 10 mM p -nitrophenylphosphate, and 0.2 μM HD-Pnk was incubated at 37°C. Aliquots (25 μl, containing 250 nmol of input p -nitrophenylphosphate) were withdrawn at the times specified and quenched immediately with <t>EDTA.</t> p -Nitrophenol formation is plotted as a function of time. (F) p -Nitrophenol concentration dependence. Reaction mixtures containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 0.2 μM HD-Pnk, and 0.625, 1.25, 2.5, 5 or 10 mM p -nitrophenylphosphate were incubated at 37°C. Aliquots (25 μl) were withdrawn at 1, 2, 5, and 10 min and quenched immediately with EDTA. p -Nitrophenol formation was plotted as a function of time, and the initial rate (nmol min −1 ) at each substrate concentration was calculated by linear regression in Prism. The averages ± the SEM of initial rates ( V ) from three separate experiments are plotted as a function of p -nitrophenol concentration. The data were fit to the Michaelis-Menten equation by nonlinear regression in Prism.
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    HD-Pnk is a copper-dependent p -nitrophenylphosphatase. (A) Metal specificity. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 10 mM (250 nmol) p -nitrophenylphosphate, 10 pmol (0.4 μM) of HD-Pnk, and either no added metal cofactor (lane –) or 1 mM the indicated divalent cation (added as chloride salt) were incubated at 37°C for 10 min. The extents of p -nitrophenol product formation are plotted in bar graph format. Each datum is the average from three separate experiments ± the standard errors of the mean (SEM). (B) Metal concentration dependence. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 10 mM (250 nmol) p -nitrophenylphosphate, 20 pmol (0.8 μM) of HD-Pnk, and 0, 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, or 5 mM CuCl 2 or NiCl 2 as indicated were incubated at 37°C for 10 min. p -Nitrophenol formation is plotted as a function of divalent cation concentration. (C) His73 is essential for p -nitrophenylphosphatase activity. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 10 mM (250 nmol) p -nitrophenylphosphate, and 10 pmol (0.4 μM) wild-type (WT) HD-Pnk or mutants H73A or D254A as specified were incubated at 37°C for 10 min. (D) Enzyme titration. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 10 mM (250 nmol) p -nitrophenylphosphate, and 0, 1. 2.5, 5, 7.5, 10, or 20 pmol of HD-Pnk were incubated at 37°C for 10 min. p -Nitrophenol formation is plotted as a function of input enzyme. Each datum is the average from three separate experiments ± the SEM. (E) Time course. A reaction mixture (250 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 10 mM p -nitrophenylphosphate, and 0.2 μM HD-Pnk was incubated at 37°C. Aliquots (25 μl, containing 250 nmol of input p -nitrophenylphosphate) were withdrawn at the times specified and quenched immediately with EDTA. p -Nitrophenol formation is plotted as a function of time. (F) p -Nitrophenol concentration dependence. Reaction mixtures containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 0.2 μM HD-Pnk, and 0.625, 1.25, 2.5, 5 or 10 mM p -nitrophenylphosphate were incubated at 37°C. Aliquots (25 μl) were withdrawn at 1, 2, 5, and 10 min and quenched immediately with EDTA. p -Nitrophenol formation was plotted as a function of time, and the initial rate (nmol min −1 ) at each substrate concentration was calculated by linear regression in Prism. The averages ± the SEM of initial rates ( V ) from three separate experiments are plotted as a function of p -nitrophenol concentration. The data were fit to the Michaelis-Menten equation by nonlinear regression in Prism.

    Journal: Journal of Bacteriology

    Article Title: Structure-Function Analysis of the Phosphoesterase Component of the Nucleic Acid End-Healing Enzyme Runella slithyformis HD-Pnk

    doi: 10.1128/JB.00292-19

    Figure Lengend Snippet: HD-Pnk is a copper-dependent p -nitrophenylphosphatase. (A) Metal specificity. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 10 mM (250 nmol) p -nitrophenylphosphate, 10 pmol (0.4 μM) of HD-Pnk, and either no added metal cofactor (lane –) or 1 mM the indicated divalent cation (added as chloride salt) were incubated at 37°C for 10 min. The extents of p -nitrophenol product formation are plotted in bar graph format. Each datum is the average from three separate experiments ± the standard errors of the mean (SEM). (B) Metal concentration dependence. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 10 mM (250 nmol) p -nitrophenylphosphate, 20 pmol (0.8 μM) of HD-Pnk, and 0, 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, or 5 mM CuCl 2 or NiCl 2 as indicated were incubated at 37°C for 10 min. p -Nitrophenol formation is plotted as a function of divalent cation concentration. (C) His73 is essential for p -nitrophenylphosphatase activity. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 10 mM (250 nmol) p -nitrophenylphosphate, and 10 pmol (0.4 μM) wild-type (WT) HD-Pnk or mutants H73A or D254A as specified were incubated at 37°C for 10 min. (D) Enzyme titration. Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 10 mM (250 nmol) p -nitrophenylphosphate, and 0, 1. 2.5, 5, 7.5, 10, or 20 pmol of HD-Pnk were incubated at 37°C for 10 min. p -Nitrophenol formation is plotted as a function of input enzyme. Each datum is the average from three separate experiments ± the SEM. (E) Time course. A reaction mixture (250 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 10 mM p -nitrophenylphosphate, and 0.2 μM HD-Pnk was incubated at 37°C. Aliquots (25 μl, containing 250 nmol of input p -nitrophenylphosphate) were withdrawn at the times specified and quenched immediately with EDTA. p -Nitrophenol formation is plotted as a function of time. (F) p -Nitrophenol concentration dependence. Reaction mixtures containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 0.2 μM HD-Pnk, and 0.625, 1.25, 2.5, 5 or 10 mM p -nitrophenylphosphate were incubated at 37°C. Aliquots (25 μl) were withdrawn at 1, 2, 5, and 10 min and quenched immediately with EDTA. p -Nitrophenol formation was plotted as a function of time, and the initial rate (nmol min −1 ) at each substrate concentration was calculated by linear regression in Prism. The averages ± the SEM of initial rates ( V ) from three separate experiments are plotted as a function of p -nitrophenol concentration. The data were fit to the Michaelis-Menten equation by nonlinear regression in Prism.

    Article Snippet: The products were analyzed by electrophoresis at 55 W constant power through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate–1 mM EDTA, visualized by scanning the gel with a FujiFilm FLA-7000 imager, and quantified using ImageJ software.

    Techniques: Incubation, Concentration Assay, Activity Assay, Titration

    Formation of a novel RNA product during Tpt1 reaction with 2′-phosphate RNA. ( A ) Reaction mixtures (10 μl) containing 100 mM Tris–HCl, pH 7.5, 0.2 μM (2 pmol) 5′ 32 P-labeled 6-mer 2′-PO 4 RNA (shown at bottom), 1 mM NAD + , and 0.5 μM (5 pmol) Tpt1 from the sources specified were incubated at 37°C for 30 min. Tpt1 was omitted from the control reaction mixture in lane –. The sources of enzyme are as follows: Runella slithyformis , Rsl; Clostridium thermocellum , Clth; Chaetomium themophilum , Chth; Homo sapiens , Hsa; Aeropyrum pernix , Ape, Pyrococcus horikoshii , Pho; and Archaeoglobus fulgidus , Afu. The step 2-defective RslTpt1-R64A mutant was assayed in parallel (leftmost lane) in order to demarcate the 2′-P-ADPR RNA intermediate in the canonical Tpt1 reaction pathway. The reactions were quenched by adding three volumes of cold 90% formamide, 50 mM EDTA. ( B ) A reaction mixture containing 100 mM Tris–HCl, pH 7.5, 0.2 μM 5′ 32 P-labeled 6-mer 2′-PO 4 RNA, 1 mM NAD + , and 0.5 μM (5 pmol) ApeTpt1 was incubated at 37°C. Aliquots (10 μl) were withdrawn at the times specified and quenched immediately with three volumes of cold 90% formamide, 50 mM EDTA. The time 0 sample was withdrawn prior to adding ApeTpt1. The reaction products were analyzed by urea-PAGE and visualized by autoradiography. The positions of the 6-mer 2′-PO 4 RNA substrate (2′-P), 2′-P-ADPR RNA intermediate, and 2′-OH RNA product (2′-OH) of the canonical two-step Tpt1 reaction are indicated on the left and right. The novel RNA product formed by some of the Tpt1 enzymes is denoted by an asterisk. The yields of 2′-OH RNA and novel RNA (expressed as percent of total labeled RNA) are indicated below the lanes.

    Journal: Nucleic Acids Research

    Article Title: NAD+-dependent synthesis of a 5′-phospho-ADP-ribosylated RNA/DNA cap by RNA 2′-phosphotransferase Tpt1

    doi: 10.1093/nar/gky792

    Figure Lengend Snippet: Formation of a novel RNA product during Tpt1 reaction with 2′-phosphate RNA. ( A ) Reaction mixtures (10 μl) containing 100 mM Tris–HCl, pH 7.5, 0.2 μM (2 pmol) 5′ 32 P-labeled 6-mer 2′-PO 4 RNA (shown at bottom), 1 mM NAD + , and 0.5 μM (5 pmol) Tpt1 from the sources specified were incubated at 37°C for 30 min. Tpt1 was omitted from the control reaction mixture in lane –. The sources of enzyme are as follows: Runella slithyformis , Rsl; Clostridium thermocellum , Clth; Chaetomium themophilum , Chth; Homo sapiens , Hsa; Aeropyrum pernix , Ape, Pyrococcus horikoshii , Pho; and Archaeoglobus fulgidus , Afu. The step 2-defective RslTpt1-R64A mutant was assayed in parallel (leftmost lane) in order to demarcate the 2′-P-ADPR RNA intermediate in the canonical Tpt1 reaction pathway. The reactions were quenched by adding three volumes of cold 90% formamide, 50 mM EDTA. ( B ) A reaction mixture containing 100 mM Tris–HCl, pH 7.5, 0.2 μM 5′ 32 P-labeled 6-mer 2′-PO 4 RNA, 1 mM NAD + , and 0.5 μM (5 pmol) ApeTpt1 was incubated at 37°C. Aliquots (10 μl) were withdrawn at the times specified and quenched immediately with three volumes of cold 90% formamide, 50 mM EDTA. The time 0 sample was withdrawn prior to adding ApeTpt1. The reaction products were analyzed by urea-PAGE and visualized by autoradiography. The positions of the 6-mer 2′-PO 4 RNA substrate (2′-P), 2′-P-ADPR RNA intermediate, and 2′-OH RNA product (2′-OH) of the canonical two-step Tpt1 reaction are indicated on the left and right. The novel RNA product formed by some of the Tpt1 enzymes is denoted by an asterisk. The yields of 2′-OH RNA and novel RNA (expressed as percent of total labeled RNA) are indicated below the lanes.

    Article Snippet: The products were analyzed by electrophoresis (at 55 W constant power) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA and visualized by autoradiography and/or scanning the gel with a Fujifilm FLA-7000 imaging device.

    Techniques: Labeling, Incubation, Mutagenesis, Polyacrylamide Gel Electrophoresis, Autoradiography

    ApeTpt1 synthesizes a phosphatase-resistant 5′ cap structure. Reaction mixtures (10 μl) containing 100 mM Tris–HCl, pH 7.5, 0.2 μM (2 pmol) 5′ 32 P-labeled 6-mer 2′-PO 4 RNA (shown at bottom), 1 mM NAD + , and 0.5 μM (5 pmol) ApeTpt1 (where indicated by +) were incubated at 37°C for 60 min. The reaction mixtures were heated at 65°C for 5 min and then either treated for 10 min at 37°C with 10 U of calf intestine alkaline phosphatase (CIP; from NEB) in 1× Cutsmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 100 μg/ml BSA, pH 7.9) or mock-incubated without phosphatase. The reactions were quenched with three volumes of cold 90% formamide, 50 mM EDTA and the products were analyzed by urea-PAGE and visualized by autoradiography.

    Journal: Nucleic Acids Research

    Article Title: NAD+-dependent synthesis of a 5′-phospho-ADP-ribosylated RNA/DNA cap by RNA 2′-phosphotransferase Tpt1

    doi: 10.1093/nar/gky792

    Figure Lengend Snippet: ApeTpt1 synthesizes a phosphatase-resistant 5′ cap structure. Reaction mixtures (10 μl) containing 100 mM Tris–HCl, pH 7.5, 0.2 μM (2 pmol) 5′ 32 P-labeled 6-mer 2′-PO 4 RNA (shown at bottom), 1 mM NAD + , and 0.5 μM (5 pmol) ApeTpt1 (where indicated by +) were incubated at 37°C for 60 min. The reaction mixtures were heated at 65°C for 5 min and then either treated for 10 min at 37°C with 10 U of calf intestine alkaline phosphatase (CIP; from NEB) in 1× Cutsmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 100 μg/ml BSA, pH 7.9) or mock-incubated without phosphatase. The reactions were quenched with three volumes of cold 90% formamide, 50 mM EDTA and the products were analyzed by urea-PAGE and visualized by autoradiography.

    Article Snippet: The products were analyzed by electrophoresis (at 55 W constant power) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA and visualized by autoradiography and/or scanning the gel with a Fujifilm FLA-7000 imaging device.

    Techniques: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

    RNA 2′- and 3′-phosphomonoesterase activities of HD-Pnk. (A) Enzyme titration. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM NiCl 2 , 20 nM (0.2 pmol) 32 P-labeled 10-mer RNAs with 2′-phosphate or 3′-phosphate ends (depicted at the bottom, with the labeled phosphate indicated by a dot), and increasing amounts of wild-type HD-Pnk, as specified, were incubated at 37°C for 10 min. The reactions were quenched with formamide-EDTA, and the products were analyzed by urea-PAGE, along with an otherwise identical 10-mer RNA with a 2′,3′-cyclic phosphate end (2′,3′ > p). An autoradiogram of the gel is shown. (B) Divalent cation requirements. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 20 nM (0.2 pmol) 32 P-labeled 10-mer HO RNA3′p, 0.25 pmol HD-Pnk, and either no divalent cation (−) or 1 mM MgCl 2 , MnCl 2 , NiCl 2 , CaCl 2 , CoCl 2 , ZnCl 2 , or CuCl 2 , as indicated, were incubated at 37°C for 10 min. The reactions were quenched with formamide-EDTA, and the products were analyzed by urea-PAGE. An autoradiogram of the gel is shown. (C) Kinetics. Reaction mixtures (80 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM NiCl 2 , 20 nM (1.6 pmol) 32 P-labeled 10-mer HO RNA3′p or HO RNA2′p substrate, and 40 pmol HD-Pnk were incubated at 37°C. Aliquots (10 μl) were withdrawn at the times indicated on the x axis and were quenched immediately with formamide-EDTA. The time zero sample was taken prior to the addition of HD-Pnk. The extent of formation of HO RNA OH is plotted as a function of the reaction time. Each datum in the graph is the average ± SEM from three independent experiments.

    Journal: Journal of Bacteriology

    Article Title: Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

    doi: 10.1128/JB.00739-16

    Figure Lengend Snippet: RNA 2′- and 3′-phosphomonoesterase activities of HD-Pnk. (A) Enzyme titration. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM NiCl 2 , 20 nM (0.2 pmol) 32 P-labeled 10-mer RNAs with 2′-phosphate or 3′-phosphate ends (depicted at the bottom, with the labeled phosphate indicated by a dot), and increasing amounts of wild-type HD-Pnk, as specified, were incubated at 37°C for 10 min. The reactions were quenched with formamide-EDTA, and the products were analyzed by urea-PAGE, along with an otherwise identical 10-mer RNA with a 2′,3′-cyclic phosphate end (2′,3′ > p). An autoradiogram of the gel is shown. (B) Divalent cation requirements. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 20 nM (0.2 pmol) 32 P-labeled 10-mer HO RNA3′p, 0.25 pmol HD-Pnk, and either no divalent cation (−) or 1 mM MgCl 2 , MnCl 2 , NiCl 2 , CaCl 2 , CoCl 2 , ZnCl 2 , or CuCl 2 , as indicated, were incubated at 37°C for 10 min. The reactions were quenched with formamide-EDTA, and the products were analyzed by urea-PAGE. An autoradiogram of the gel is shown. (C) Kinetics. Reaction mixtures (80 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM NiCl 2 , 20 nM (1.6 pmol) 32 P-labeled 10-mer HO RNA3′p or HO RNA2′p substrate, and 40 pmol HD-Pnk were incubated at 37°C. Aliquots (10 μl) were withdrawn at the times indicated on the x axis and were quenched immediately with formamide-EDTA. The time zero sample was taken prior to the addition of HD-Pnk. The extent of formation of HO RNA OH is plotted as a function of the reaction time. Each datum in the graph is the average ± SEM from three independent experiments.

    Article Snippet: The products were analyzed by electrophoresis (at a constant power of 55 W) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA, visualized by autoradiography, and quantified by scanning the gel with a Fujifilm FLA-7000 imager.

    Techniques: Titration, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

    NTP donor requirements for the HD-Pnk kinase. (A) Kinase assay. A reaction mixture (60 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM MgCl 2 , 2.5 mM DTT, 100 μM ATP, 20 nM 3′- 32 P-labeled 21-mer HO DNA (depicted at the bottom, with the labeled phosphate indicated by a dot), and 0.5 μM HD-Pnk was incubated at 22°C. Aliquots (10 μl) were withdrawn at 0.5, 1, 2, and 3.5 min and quenched immediately with formamide-EDTA. The time zero sample was taken prior to the addition of HD-Pnk. The products were analyzed by urea-PAGE and visualized by autoradiography. The positions of the 5′-OH DNA substrate and the 5′-PO 4 DNA product are indicated. (B) NTP substrate specificity. Kinase reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM MgCl 2 , 2.5 mM DTT, 0.2 pmol (20 nM) 3′- 32 P-labeled 21-mer HO DNA, 0.25 μM (2.5 pmol) HD-Pnk, and 0, 1, 10, 100 or 1,000 μM ATP, GTP, CTP, UTP, or dATP, as specified, were incubated at 22°C for 5 min. The products were analyzed by urea-PAGE. The extents of 5′ phosphorylation were quantified by scanning the gel and are plotted as a function of the NTP or dNTP concentration. Each datum in the graph is the average ± SEM from three separate experiments.

    Journal: Journal of Bacteriology

    Article Title: Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

    doi: 10.1128/JB.00739-16

    Figure Lengend Snippet: NTP donor requirements for the HD-Pnk kinase. (A) Kinase assay. A reaction mixture (60 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM MgCl 2 , 2.5 mM DTT, 100 μM ATP, 20 nM 3′- 32 P-labeled 21-mer HO DNA (depicted at the bottom, with the labeled phosphate indicated by a dot), and 0.5 μM HD-Pnk was incubated at 22°C. Aliquots (10 μl) were withdrawn at 0.5, 1, 2, and 3.5 min and quenched immediately with formamide-EDTA. The time zero sample was taken prior to the addition of HD-Pnk. The products were analyzed by urea-PAGE and visualized by autoradiography. The positions of the 5′-OH DNA substrate and the 5′-PO 4 DNA product are indicated. (B) NTP substrate specificity. Kinase reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM MgCl 2 , 2.5 mM DTT, 0.2 pmol (20 nM) 3′- 32 P-labeled 21-mer HO DNA, 0.25 μM (2.5 pmol) HD-Pnk, and 0, 1, 10, 100 or 1,000 μM ATP, GTP, CTP, UTP, or dATP, as specified, were incubated at 22°C for 5 min. The products were analyzed by urea-PAGE. The extents of 5′ phosphorylation were quantified by scanning the gel and are plotted as a function of the NTP or dNTP concentration. Each datum in the graph is the average ± SEM from three separate experiments.

    Article Snippet: The products were analyzed by electrophoresis (at a constant power of 55 W) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA, visualized by autoradiography, and quantified by scanning the gel with a Fujifilm FLA-7000 imager.

    Techniques: Kinase Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography, Concentration Assay

    DNA 3′-phosphatase activity of HD-Pnk. (A) Divalent cation requirements. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 0.1 μM (1 pmol) 5′- 32 P-labeled 10-mer pDNAp (depicted at the bottom, with the labeled phosphate indicated by a dot), 5 pmol HD-Pnk, and either no divalent cation (−) or 1 mM MgCl 2 , MnCl 2 , NiCl 2 , CaCl 2 , CoCl 2 , CuCl 2 , or ZnCl 2 , as indicated, were incubated at 37°C for 10 min. (B) Effects of mutations. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 0.1 μM (1 pmol) 5′- 32 P-labeled 10-mer pDNAp, and 5 pmol wild-type or mutant HD-Pnk, as specified, were incubated at 37°C for 10 min. HD-Pnk was omitted from the control reaction in lane –. The reactions shown in panels A and B were quenched with formamide-EDTA, and the products were analyzed by urea-PAGE. Autoradiograms of the gels are shown. (C) Enzyme titration. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 0.1 μM (1 pmol) 5′- 32 P-labeled 10-mer pDNAp, either 1 mM CuCl 2 or 1 mM NiCl 2 , and increasing amounts of wild-type HD-Pnk, as specified, were incubated at 37°C for 10 min. The extent of formation of pDNA OH is plotted as a function of the input enzyme amount. Each datum in the graph is the average ± SEM from three independent experiments. (D) Kinetics. Reaction mixtures (80 μl) containing 100 mM Tris-acetate (pH 6.0), either 1 mM CuCl 2 or 1 mM NiCl 2 , 0.1 μM (8 pmol) 5′- 32 P-labeled 10-mer pDNAp, and 40 pmol wild-type HD-Pnk were incubated at 37°C. Aliquots (10 μl) were withdrawn at the times specified on the x axis. The extent of formation of pDNA OH is plotted as a function of time. Each datum in the graph is the average ± SEM from three independent experiments.

    Journal: Journal of Bacteriology

    Article Title: Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

    doi: 10.1128/JB.00739-16

    Figure Lengend Snippet: DNA 3′-phosphatase activity of HD-Pnk. (A) Divalent cation requirements. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 0.1 μM (1 pmol) 5′- 32 P-labeled 10-mer pDNAp (depicted at the bottom, with the labeled phosphate indicated by a dot), 5 pmol HD-Pnk, and either no divalent cation (−) or 1 mM MgCl 2 , MnCl 2 , NiCl 2 , CaCl 2 , CoCl 2 , CuCl 2 , or ZnCl 2 , as indicated, were incubated at 37°C for 10 min. (B) Effects of mutations. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl 2 , 0.1 μM (1 pmol) 5′- 32 P-labeled 10-mer pDNAp, and 5 pmol wild-type or mutant HD-Pnk, as specified, were incubated at 37°C for 10 min. HD-Pnk was omitted from the control reaction in lane –. The reactions shown in panels A and B were quenched with formamide-EDTA, and the products were analyzed by urea-PAGE. Autoradiograms of the gels are shown. (C) Enzyme titration. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 0.1 μM (1 pmol) 5′- 32 P-labeled 10-mer pDNAp, either 1 mM CuCl 2 or 1 mM NiCl 2 , and increasing amounts of wild-type HD-Pnk, as specified, were incubated at 37°C for 10 min. The extent of formation of pDNA OH is plotted as a function of the input enzyme amount. Each datum in the graph is the average ± SEM from three independent experiments. (D) Kinetics. Reaction mixtures (80 μl) containing 100 mM Tris-acetate (pH 6.0), either 1 mM CuCl 2 or 1 mM NiCl 2 , 0.1 μM (8 pmol) 5′- 32 P-labeled 10-mer pDNAp, and 40 pmol wild-type HD-Pnk were incubated at 37°C. Aliquots (10 μl) were withdrawn at the times specified on the x axis. The extent of formation of pDNA OH is plotted as a function of time. Each datum in the graph is the average ± SEM from three independent experiments.

    Article Snippet: The products were analyzed by electrophoresis (at a constant power of 55 W) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA, visualized by autoradiography, and quantified by scanning the gel with a Fujifilm FLA-7000 imager.

    Techniques: Activity Assay, Labeling, Incubation, Mutagenesis, Polyacrylamide Gel Electrophoresis, Titration

    RNA 2′,3′-cyclic phosphodiesterase activity of HD-Pnk. (A) CPDase assay. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM NiCl 2 , 20 nM (0.2 pmol) 32 P-labeled 10-mer HO RNA > p (depicted at the bottom, with the labeled phosphate indicated by a dot), and 5 pmol wild-type or mutant HD-Pnk, as specified, or no added enzyme (−) were incubated at 37°C for 30 min. A control T4 Pnkp reaction mixture containing 70 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 20 nM (0.2 pmol) 32 P-labeled 10-mer HO RNA > p, and 5 pmol T4 Pnkp was incubated at 37°C for 30 min. The reactions were quenched with an equal volume of formamide-EDTA, and the products were analyzed by urea-PAGE. An autoradiogram of the gel is shown. The states of the 3′ ends of the radiolabeled RNAs are indicated at the right. (B) Divalent cation requirements. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 20 nM (0.2 pmol) 32 P-labeled 10-mer HO RNA > p, 5 pmol wild-type HD-Pnk, and either no divalent cation (−) plus 10 mM EDTA (+EDTA), no divalent cation (−), or 5 mM MgCl 2 , MnCl 2 , NiCl 2 , CaCl 2 , CoCl 2 , ZnCl 2 , or CuCl 2 , as indicated, were incubated at 37°C for 10 min. The reactions were quenched with formamide-EDTA, and the products were analyzed by urea-PAGE. An autoradiogram of the gel is shown. (C) Kinetic profile of the CPDase reaction. Reaction mixtures (80 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM NiCl 2 , 20 nM (1.6 pmol) 32 P-labeled 10-mer HO RNA > p, and 40 pmol wild-type HD-Pnk were incubated at 37°C. Aliquots (10 μl) were withdrawn at 1, 3, 5, 10, 15, and 30 min and quenched immediately with formamide-EDTA. The time zero sample was taken prior to the addition of HD-Pnk. The products were analyzed by urea-PAGE. The extent of conversion of HO RNA > p to HO RNAp and HO RNA OH was quantified by scanning the gel, and results are plotted as a function of time. (D) Enzyme titration. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM NiCl 2 , 20 nM (0.2 pmol) 32 P-labeled 10-mer HO RNA > p, and increasing amounts of wild-type HD-Pnk or mutant H73A, as specified, were incubated at 37°C for 10 min. The reactions were quenched with formamide-EDTA, and the products were analyzed by urea-PAGE. An autoradiogram of the gel is shown.

    Journal: Journal of Bacteriology

    Article Title: Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

    doi: 10.1128/JB.00739-16

    Figure Lengend Snippet: RNA 2′,3′-cyclic phosphodiesterase activity of HD-Pnk. (A) CPDase assay. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM NiCl 2 , 20 nM (0.2 pmol) 32 P-labeled 10-mer HO RNA > p (depicted at the bottom, with the labeled phosphate indicated by a dot), and 5 pmol wild-type or mutant HD-Pnk, as specified, or no added enzyme (−) were incubated at 37°C for 30 min. A control T4 Pnkp reaction mixture containing 70 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 20 nM (0.2 pmol) 32 P-labeled 10-mer HO RNA > p, and 5 pmol T4 Pnkp was incubated at 37°C for 30 min. The reactions were quenched with an equal volume of formamide-EDTA, and the products were analyzed by urea-PAGE. An autoradiogram of the gel is shown. The states of the 3′ ends of the radiolabeled RNAs are indicated at the right. (B) Divalent cation requirements. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 20 nM (0.2 pmol) 32 P-labeled 10-mer HO RNA > p, 5 pmol wild-type HD-Pnk, and either no divalent cation (−) plus 10 mM EDTA (+EDTA), no divalent cation (−), or 5 mM MgCl 2 , MnCl 2 , NiCl 2 , CaCl 2 , CoCl 2 , ZnCl 2 , or CuCl 2 , as indicated, were incubated at 37°C for 10 min. The reactions were quenched with formamide-EDTA, and the products were analyzed by urea-PAGE. An autoradiogram of the gel is shown. (C) Kinetic profile of the CPDase reaction. Reaction mixtures (80 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM NiCl 2 , 20 nM (1.6 pmol) 32 P-labeled 10-mer HO RNA > p, and 40 pmol wild-type HD-Pnk were incubated at 37°C. Aliquots (10 μl) were withdrawn at 1, 3, 5, 10, 15, and 30 min and quenched immediately with formamide-EDTA. The time zero sample was taken prior to the addition of HD-Pnk. The products were analyzed by urea-PAGE. The extent of conversion of HO RNA > p to HO RNAp and HO RNA OH was quantified by scanning the gel, and results are plotted as a function of time. (D) Enzyme titration. Reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 5 mM NiCl 2 , 20 nM (0.2 pmol) 32 P-labeled 10-mer HO RNA > p, and increasing amounts of wild-type HD-Pnk or mutant H73A, as specified, were incubated at 37°C for 10 min. The reactions were quenched with formamide-EDTA, and the products were analyzed by urea-PAGE. An autoradiogram of the gel is shown.

    Article Snippet: The products were analyzed by electrophoresis (at a constant power of 55 W) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA, visualized by autoradiography, and quantified by scanning the gel with a Fujifilm FLA-7000 imager.

    Techniques: Activity Assay, Labeling, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, Titration

    Formation of a novel RNA product during Tpt1 reaction with 2′-phosphate RNA. ( A ) Reaction mixtures (10 μl) containing 100 mM Tris–HCl, pH 7.5, 0.2 μM (2 pmol) 5′ 32 P-labeled 6-mer 2′-PO 4 RNA (shown at bottom), 1 mM NAD + , and 0.5 μM (5 pmol) Tpt1 from the sources specified were incubated at 37°C for 30 min. Tpt1 was omitted from the control reaction mixture in lane –. The sources of enzyme are as follows: Runella slithyformis , Rsl; Clostridium thermocellum , Clth; Chaetomium themophilum , Chth; Homo sapiens , Hsa; Aeropyrum pernix , Ape, Pyrococcus horikoshii , Pho; and Archaeoglobus fulgidus , Afu. The step 2-defective RslTpt1-R64A mutant was assayed in parallel (leftmost lane) in order to demarcate the 2′-P-ADPR RNA intermediate in the canonical Tpt1 reaction pathway. The reactions were quenched by adding three volumes of cold 90% formamide, 50 mM EDTA. ( B ) A reaction mixture containing 100 mM Tris–HCl, pH 7.5, 0.2 μM 5′ 32 P-labeled 6-mer 2′-PO 4 RNA, 1 mM NAD + , and 0.5 μM (5 pmol) ApeTpt1 was incubated at 37°C. Aliquots (10 μl) were withdrawn at the times specified and quenched immediately with three volumes of cold 90% formamide, 50 mM EDTA. The time 0 sample was withdrawn prior to adding ApeTpt1. The reaction products were analyzed by urea-PAGE and visualized by autoradiography. The positions of the 6-mer 2′-PO 4 RNA substrate (2′-P), 2′-P-ADPR RNA intermediate, and 2′-OH RNA product (2′-OH) of the canonical two-step Tpt1 reaction are indicated on the left and right. The novel RNA product formed by some of the Tpt1 enzymes is denoted by an asterisk. The yields of 2′-OH RNA and novel RNA (expressed as percent of total labeled RNA) are indicated below the lanes.

    Journal: Nucleic Acids Research

    Article Title: NAD+-dependent synthesis of a 5′-phospho-ADP-ribosylated RNA/DNA cap by RNA 2′-phosphotransferase Tpt1

    doi: 10.1093/nar/gky792

    Figure Lengend Snippet: Formation of a novel RNA product during Tpt1 reaction with 2′-phosphate RNA. ( A ) Reaction mixtures (10 μl) containing 100 mM Tris–HCl, pH 7.5, 0.2 μM (2 pmol) 5′ 32 P-labeled 6-mer 2′-PO 4 RNA (shown at bottom), 1 mM NAD + , and 0.5 μM (5 pmol) Tpt1 from the sources specified were incubated at 37°C for 30 min. Tpt1 was omitted from the control reaction mixture in lane –. The sources of enzyme are as follows: Runella slithyformis , Rsl; Clostridium thermocellum , Clth; Chaetomium themophilum , Chth; Homo sapiens , Hsa; Aeropyrum pernix , Ape, Pyrococcus horikoshii , Pho; and Archaeoglobus fulgidus , Afu. The step 2-defective RslTpt1-R64A mutant was assayed in parallel (leftmost lane) in order to demarcate the 2′-P-ADPR RNA intermediate in the canonical Tpt1 reaction pathway. The reactions were quenched by adding three volumes of cold 90% formamide, 50 mM EDTA. ( B ) A reaction mixture containing 100 mM Tris–HCl, pH 7.5, 0.2 μM 5′ 32 P-labeled 6-mer 2′-PO 4 RNA, 1 mM NAD + , and 0.5 μM (5 pmol) ApeTpt1 was incubated at 37°C. Aliquots (10 μl) were withdrawn at the times specified and quenched immediately with three volumes of cold 90% formamide, 50 mM EDTA. The time 0 sample was withdrawn prior to adding ApeTpt1. The reaction products were analyzed by urea-PAGE and visualized by autoradiography. The positions of the 6-mer 2′-PO 4 RNA substrate (2′-P), 2′-P-ADPR RNA intermediate, and 2′-OH RNA product (2′-OH) of the canonical two-step Tpt1 reaction are indicated on the left and right. The novel RNA product formed by some of the Tpt1 enzymes is denoted by an asterisk. The yields of 2′-OH RNA and novel RNA (expressed as percent of total labeled RNA) are indicated below the lanes.

    Article Snippet: The products were analyzed by electrophoresis (at 55 W constant power) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA and visualized by autoradiography and/or scanning the gel with a Fujifilm FLA-7000 imaging device.

    Techniques: Labeling, Incubation, Mutagenesis, Polyacrylamide Gel Electrophoresis, Autoradiography

    ApeTpt1 synthesizes a phosphatase-resistant 5′ cap structure. Reaction mixtures (10 μl) containing 100 mM Tris–HCl, pH 7.5, 0.2 μM (2 pmol) 5′ 32 P-labeled 6-mer 2′-PO 4 RNA (shown at bottom), 1 mM NAD + , and 0.5 μM (5 pmol) ApeTpt1 (where indicated by +) were incubated at 37°C for 60 min. The reaction mixtures were heated at 65°C for 5 min and then either treated for 10 min at 37°C with 10 U of calf intestine alkaline phosphatase (CIP; from NEB) in 1× Cutsmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 100 μg/ml BSA, pH 7.9) or mock-incubated without phosphatase. The reactions were quenched with three volumes of cold 90% formamide, 50 mM EDTA and the products were analyzed by urea-PAGE and visualized by autoradiography.

    Journal: Nucleic Acids Research

    Article Title: NAD+-dependent synthesis of a 5′-phospho-ADP-ribosylated RNA/DNA cap by RNA 2′-phosphotransferase Tpt1

    doi: 10.1093/nar/gky792

    Figure Lengend Snippet: ApeTpt1 synthesizes a phosphatase-resistant 5′ cap structure. Reaction mixtures (10 μl) containing 100 mM Tris–HCl, pH 7.5, 0.2 μM (2 pmol) 5′ 32 P-labeled 6-mer 2′-PO 4 RNA (shown at bottom), 1 mM NAD + , and 0.5 μM (5 pmol) ApeTpt1 (where indicated by +) were incubated at 37°C for 60 min. The reaction mixtures were heated at 65°C for 5 min and then either treated for 10 min at 37°C with 10 U of calf intestine alkaline phosphatase (CIP; from NEB) in 1× Cutsmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 100 μg/ml BSA, pH 7.9) or mock-incubated without phosphatase. The reactions were quenched with three volumes of cold 90% formamide, 50 mM EDTA and the products were analyzed by urea-PAGE and visualized by autoradiography.

    Article Snippet: The products were analyzed by electrophoresis (at 55 W constant power) through a 40-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA and visualized by autoradiography and/or scanning the gel with a Fujifilm FLA-7000 imaging device.

    Techniques: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography