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    Structured Review

    Millipore polyethyleneimine cellulose plates
    Polyethyleneimine Cellulose Plates, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 10 article reviews
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    polyethyleneimine cellulose plates - by Bioz Stars, 2020-04
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    Related Articles

    Centrifugation:

    Article Title: The Bifunctional Enzyme SpoT Is Involved in the Clarithromycin Tolerance of Helicobacter pylori by Upregulating the Transporters HP0939, HP1017, HP0497, and HP0471
    Article Snippet: Briefly, H. pylori strains were grown overnight in brucella broth containing 10% FBS to the early exponential phase (OD600 , approximately 0.4), diluted back to an OD600 of 0.2, and incubated for an additional 2 h. When all strains reached an OD600 of approximately 0.3, samples (OD600 , approximately 0.3) of each culture were removed and pelleted via centrifugation at 10,000 rpm for 5 min, after which samples were resuspended in 250 μl of liquid culture medium. .. The acid extracts were centrifuged briefly, and the supernatant fluids were spotted onto polyethyleneimine-cellulose plates (Sigma-Aldrich), dried, and developed in 1.5 M KH2 PO4 (pH 3.4) for approximately 2.5 h. The results were obtained using phosphor screen scanning (Bio-Rad).

    Article Title: The Putative "Switch 2" Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein
    Article Snippet: After a 30-min incubation, the charcoal was removed by centrifugation at 10,000 × g for 2 min, and immune complexes were collected by incubation for 1 h with protein A-Sepharose beads coupled to goat anti-mouse IgG. .. A 5-μl aliquot of each eluate was subjected to TLC on polyethyleneimine cellulose plates (Sigma, St. Louis, MO) developed in 0.75 M KH2 PO4 (pH 3.4).

    Synthesized:

    Article Title: Copper Ions Stimulate Polyphosphate Degradation and Phosphate Efflux in Acidithiobacillus ferrooxidans
    Article Snippet: After incubation of the mixture at 30°C for 60 min, the reaction was stopped by loading the mixture in polyethyleneimine-cellulose plates for thin-layer chromatography (Aldrich) and development in 0.75 M KH2 PO4 , pH 3.5. .. [33 P]polyP750 was synthesized in vitro by the method of Ault-Riché et al. ( ) as described in Cardona et al. ( ).

    Autoradiography:

    Article Title: The Role of Inorganic Pyrophosphate in Aortic Valve Calcification
    Article Snippet: Samples of the medium were spotted onto polyethyleneimine-cellulose plates (Sigma-Aldrich), followed by development with 650 mM KH2 PO4 (pH 3). .. Spots identified by autoradiography were scraped into distilled water and counted as Cerenkov radiation by liquid scintillation.

    Article Title: Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages *
    Article Snippet: EDTA was added to a final concentration of 50 m m to stop the reaction, and the ATP hydrolysis products were separated by thin layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma-Aldrich). .. The plates were air-dried for autoradiography and phosphorimaging (Storm 820 PhosphorImager, GE Healthcare).

    Construct:

    Article Title: The Putative "Switch 2" Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein
    Article Snippet: A 5-μl aliquot of each eluate was subjected to TLC on polyethyleneimine cellulose plates (Sigma, St. Louis, MO) developed in 0.75 M KH2 PO4 (pH 3.4). .. Since the intrinsic GTPase activities of the various Myc-Rab1B constructs at 4°C were unknown, no attempt was made to correct for GTP hydrolysis that might have occurred during the immunoprecipitation procedure.

    Incubation:

    Article Title: The Bifunctional Enzyme SpoT Is Involved in the Clarithromycin Tolerance of Helicobacter pylori by Upregulating the Transporters HP0939, HP1017, HP0497, and HP0471
    Article Snippet: Briefly, H. pylori strains were grown overnight in brucella broth containing 10% FBS to the early exponential phase (OD600 , approximately 0.4), diluted back to an OD600 of 0.2, and incubated for an additional 2 h. When all strains reached an OD600 of approximately 0.3, samples (OD600 , approximately 0.3) of each culture were removed and pelleted via centrifugation at 10,000 rpm for 5 min, after which samples were resuspended in 250 μl of liquid culture medium. .. The acid extracts were centrifuged briefly, and the supernatant fluids were spotted onto polyethyleneimine-cellulose plates (Sigma-Aldrich), dried, and developed in 1.5 M KH2 PO4 (pH 3.4) for approximately 2.5 h. The results were obtained using phosphor screen scanning (Bio-Rad).

    Article Title: Copper Ions Stimulate Polyphosphate Degradation and Phosphate Efflux in Acidithiobacillus ferrooxidans
    Article Snippet: .. After incubation of the mixture at 30°C for 60 min, the reaction was stopped by loading the mixture in polyethyleneimine-cellulose plates for thin-layer chromatography (Aldrich) and development in 0.75 M KH2 PO4 , pH 3.5. .. Radioactive spots corresponding to the Pi liberated by the hydrolysis of polyphosphate were visualized and quantified with a phosphor imager (Molecular Imager FX; Bio-Rad).

    Article Title: Analysis of Geminivirus AL2 and L2 Proteins Reveals a Novel AL2 Silencing Suppressor Activity
    Article Snippet: .. Extracts (250 ng) were incubated with adenosine and [γ-32 P]ATP (3,000 Ci/mmol; Perkin-Elmer) at 30°C for 20 min, and products were fractionated by thin-layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma) in 1 M glacial acetic acid. .. Radiolabeled AMP was quantitated using a phosphorimager (Molecular Imager FX; Bio-Rad).

    Article Title: The Role of Inorganic Pyrophosphate in Aortic Valve Calcification
    Article Snippet: Fresh AV leaflets were incubated in DMEM (three leaflets in 1 ml) in a 5% CO2 incubator at 37°C with or without 30 μM MLS38949, a specific inhibitor of TNAP ( ) (PubChem ID 2931238, kindly provided by Dr. Jose Luis Millan, Sanford-Burnham Biomedical Research Institute, La Jolla, CA, USA). .. Samples of the medium were spotted onto polyethyleneimine-cellulose plates (Sigma-Aldrich), followed by development with 650 mM KH2 PO4 (pH 3).

    Article Title: The Putative "Switch 2" Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein
    Article Snippet: The beads were washed five times with lysis buffer and 32 P-labeled guanine nucleotides were eluted by incubation for 20 min at 68°C in 20 μl of elution buffer (20 mM HEPES, pH 7.3, 25 mM EDTA, 2.0% wt/vol SDS, 0.5 mM GDP, 0.5 mM GTP). .. A 5-μl aliquot of each eluate was subjected to TLC on polyethyleneimine cellulose plates (Sigma, St. Louis, MO) developed in 0.75 M KH2 PO4 (pH 3.4).

    Article Title: Phosphorylation of Williams Syndrome Transcription Factor by MAPK Induces a Switching between Two Distinct Chromatin Remodeling Complexes *
    Article Snippet: .. Briefly, the 5-μl reaction mixture containing 10 m m HEPES (pH 7.6), 50 m m KCl, 0.1 m m EDTA, 2 m m MgCl2 , 0.5 m m dithiothreitol, 7.5% glycerol, 0.5% Nonidet P-40, 30 μ m cold ATP, 5 μCi of [α-32 P]ATP, 20 n m plasmid DNA, and purified WINAC complexes from the stable transformant expressing FLAG-WSTF or FLAG-WSTF-S158A mutant (as described in , A and C) was incubated at 37 °C for 30 min. Hydrolyzed ATP and ADP were separated by TLC on polyethyleneimine-cellulose plates (Sigma). .. A 1-μl aliquot of the reaction mixture was spotted onto the plate, and TLC was carried out in 0.75 m KH2 PO4 .

    Article Title: Bifunctional Enzyme SpoT Is Involved in Biofilm Formation of Helicobacter pylori with Multidrug Resistance by Upregulating Efflux Pump Hp1174 (gluP)
    Article Snippet: The biofilm bacteria were washed with PBS, diluted to an OD600 of 0.2, and incubated for an additional 2 h. When all the strains reached an OD600 of approximately 0.3, samples from each culture plate were centrifuged at 10,000 rpm for 5 min and resuspended in 250 μl of liquid culture medium. .. The acid extracts were briefly centrifuged, and the supernatants were spotted onto polyethyleneimine-cellulose plates (Sigma-Aldrich), dried, and developed in 1.5 M KH2 PO4 (pH 3.4) for approximately 2.5 h. The results were obtained under phosphor screen scanning (Bio-Rad).

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: For RNase activity assays, 20 nM of labeled 20-mer ssRNA or 500 ng 2.3-kb ssRNA substrates was incubated with different concentrations of purified protein in the RNase reaction buffer (50 mM Tris-HCl at pH 8.0, 50 mM KCl, 2 mM MgCl2 , 5 mM NaH2 PO4 , and RNase IN [1 U/μL, Promega]) at 30°C for 1 h. The reaction was stopped at the time point indicated in the figures by adding TBE-Urea sample buffer (Bio-Rad) or 10 mM proteinase K. Digest patterns of 20-mer ssRNA were resolved in 20% polyacrylamide/7 M urea gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system. .. Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl.

    Article Title: Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages *
    Article Snippet: The purified gp17s (0.2–2 μ m ), either alone or with gp16 at a gp16/gp17 molar ratio (the ratio of monomeric gp16 molecules to monomeric gp17 molecules) of 10:1, were incubated at 37 °C for 20 min in a 20-μl reaction mixture. .. EDTA was added to a final concentration of 50 m m to stop the reaction, and the ATP hydrolysis products were separated by thin layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma-Aldrich).

    Activity Assay:

    Article Title: Copper Ions Stimulate Polyphosphate Degradation and Phosphate Efflux in Acidithiobacillus ferrooxidans
    Article Snippet: Paragraph title: Assay for PPX activity. ... After incubation of the mixture at 30°C for 60 min, the reaction was stopped by loading the mixture in polyethyleneimine-cellulose plates for thin-layer chromatography (Aldrich) and development in 0.75 M KH2 PO4 , pH 3.5.

    Article Title: Analysis of Geminivirus AL2 and L2 Proteins Reveals a Novel AL2 Silencing Suppressor Activity
    Article Snippet: ADK activity was determined as described previously , using total protein extracts from individual mock-inoculated plants or from plants infected with geminiviruses or TRV vectors. .. Extracts (250 ng) were incubated with adenosine and [γ-32 P]ATP (3,000 Ci/mmol; Perkin-Elmer) at 30°C for 20 min, and products were fractionated by thin-layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma) in 1 M glacial acetic acid.

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: Paragraph title: RNA-binding and RNase activity assays ... Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl.

    Article Title: Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages *
    Article Snippet: The reaction mixture contained 0.05–2 m m unlabeled (cold) ATP and 75 n m [γ-32 P]ATP (specific activity, 3,000 Ci/mmol; GE Healthcare) in ATPase buffer (50 m m Tris-HCl, pH 7.5, 0.1 m NaCl, 5 m m MgCl2 ). .. EDTA was added to a final concentration of 50 m m to stop the reaction, and the ATP hydrolysis products were separated by thin layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma-Aldrich).

    Infection:

    Article Title: Analysis of Geminivirus AL2 and L2 Proteins Reveals a Novel AL2 Silencing Suppressor Activity
    Article Snippet: ADK activity was determined as described previously , using total protein extracts from individual mock-inoculated plants or from plants infected with geminiviruses or TRV vectors. .. Extracts (250 ng) were incubated with adenosine and [γ-32 P]ATP (3,000 Ci/mmol; Perkin-Elmer) at 30°C for 20 min, and products were fractionated by thin-layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma) in 1 M glacial acetic acid.

    Expressing:

    Article Title: Phosphorylation of Williams Syndrome Transcription Factor by MAPK Induces a Switching between Two Distinct Chromatin Remodeling Complexes *
    Article Snippet: .. Briefly, the 5-μl reaction mixture containing 10 m m HEPES (pH 7.6), 50 m m KCl, 0.1 m m EDTA, 2 m m MgCl2 , 0.5 m m dithiothreitol, 7.5% glycerol, 0.5% Nonidet P-40, 30 μ m cold ATP, 5 μCi of [α-32 P]ATP, 20 n m plasmid DNA, and purified WINAC complexes from the stable transformant expressing FLAG-WSTF or FLAG-WSTF-S158A mutant (as described in , A and C) was incubated at 37 °C for 30 min. Hydrolyzed ATP and ADP were separated by TLC on polyethyleneimine-cellulose plates (Sigma). .. A 1-μl aliquot of the reaction mixture was spotted onto the plate, and TLC was carried out in 0.75 m KH2 PO4 .

    Modification:

    Article Title: Bifunctional Enzyme SpoT Is Involved in Biofilm Formation of Helicobacter pylori with Multidrug Resistance by Upregulating Efflux Pump Hp1174 (gluP)
    Article Snippet: Detection of (p)ppGpp accumulation patterns. (p)ppGpp production was assayed according to the method described in a previous study with slight modification ( ). .. The acid extracts were briefly centrifuged, and the supernatants were spotted onto polyethyleneimine-cellulose plates (Sigma-Aldrich), dried, and developed in 1.5 M KH2 PO4 (pH 3.4) for approximately 2.5 h. The results were obtained under phosphor screen scanning (Bio-Rad).

    RNA Binding Assay:

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: Paragraph title: RNA-binding and RNase activity assays ... Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl.

    Immunoprecipitation:

    Article Title: The Putative "Switch 2" Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein
    Article Snippet: Particulate material was removed by centrifugation at 10,000 × g for 2 min and the epitope-tagged Rab1B proteins were immunoprecipitated with Myc monoclonal antibody for 1 h. To reduce the free guanine nucleotides, 100 μl of 10% (wt/vol) activated charcoal in phosphate-buffered saline were added to each sample ( ). .. A 5-μl aliquot of each eluate was subjected to TLC on polyethyleneimine cellulose plates (Sigma, St. Louis, MO) developed in 0.75 M KH2 PO4 (pH 3.4).

    Cell Culture:

    Article Title: The Putative "Switch 2" Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein
    Article Snippet: Paragraph title: Assessment of the Guanine Nucleotide State of Myc-Rab1B Expressed in Cultured Cells ... A 5-μl aliquot of each eluate was subjected to TLC on polyethyleneimine cellulose plates (Sigma, St. Louis, MO) developed in 0.75 M KH2 PO4 (pH 3.4).

    Imaging:

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: For RNase activity assays, 20 nM of labeled 20-mer ssRNA or 500 ng 2.3-kb ssRNA substrates was incubated with different concentrations of purified protein in the RNase reaction buffer (50 mM Tris-HCl at pH 8.0, 50 mM KCl, 2 mM MgCl2 , 5 mM NaH2 PO4 , and RNase IN [1 U/μL, Promega]) at 30°C for 1 h. The reaction was stopped at the time point indicated in the figures by adding TBE-Urea sample buffer (Bio-Rad) or 10 mM proteinase K. Digest patterns of 20-mer ssRNA were resolved in 20% polyacrylamide/7 M urea gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system. .. Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl.

    Recombinant:

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: For RNA-binding assays, 500 ng of 2.3-kb ssRNA substrates were incubated with different concentrations of recombinant purified protein in the RNA-binding buffer (50 mM Tris-HCl at pH 8.0, and 10 mM EDTA) on ice for 30 min. After incubation, the reaction mixture was separated on 1% agarose gels and stained with ethidium bromide. .. Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl.

    Radioactivity:

    Article Title: The Putative "Switch 2" Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein
    Article Snippet: A 5-μl aliquot of each eluate was subjected to TLC on polyethyleneimine cellulose plates (Sigma, St. Louis, MO) developed in 0.75 M KH2 PO4 (pH 3.4). .. Radioactivity in the GTP and GDP spots was quantified with a Molecular Dynamics phosphorimaging system.

    Mutagenesis:

    Article Title: Phosphorylation of Williams Syndrome Transcription Factor by MAPK Induces a Switching between Two Distinct Chromatin Remodeling Complexes *
    Article Snippet: .. Briefly, the 5-μl reaction mixture containing 10 m m HEPES (pH 7.6), 50 m m KCl, 0.1 m m EDTA, 2 m m MgCl2 , 0.5 m m dithiothreitol, 7.5% glycerol, 0.5% Nonidet P-40, 30 μ m cold ATP, 5 μCi of [α-32 P]ATP, 20 n m plasmid DNA, and purified WINAC complexes from the stable transformant expressing FLAG-WSTF or FLAG-WSTF-S158A mutant (as described in , A and C) was incubated at 37 °C for 30 min. Hydrolyzed ATP and ADP were separated by TLC on polyethyleneimine-cellulose plates (Sigma). .. A 1-μl aliquot of the reaction mixture was spotted onto the plate, and TLC was carried out in 0.75 m KH2 PO4 .

    Labeling:

    Article Title: The Bifunctional Enzyme SpoT Is Involved in the Clarithromycin Tolerance of Helicobacter pylori by Upregulating the Transporters HP0939, HP1017, HP0497, and HP0471
    Article Snippet: Subsequently, the experimental cells were treated with CLA (0.12 μg ml−1 ) for 1 h. Aliquots that were labeled for the duration of the experimental cultures were used as controls. .. The acid extracts were centrifuged briefly, and the supernatant fluids were spotted onto polyethyleneimine-cellulose plates (Sigma-Aldrich), dried, and developed in 1.5 M KH2 PO4 (pH 3.4) for approximately 2.5 h. The results were obtained using phosphor screen scanning (Bio-Rad).

    Article Title: Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3?- > 5? exonuclease containing S1 and KH RNA-binding domains
    Article Snippet: Oligo(rA)23 was labeled at the 5′-end by polynucleotide kinase and gel purified. .. Ascending thin layer chromatography on polyethyleneimine–cellulose plates (Sigma) was conducted in 1 M formic acid and 0.5 M LiCl, as described previously ( ).

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: For RNase activity assays, 20 nM of labeled 20-mer ssRNA or 500 ng 2.3-kb ssRNA substrates was incubated with different concentrations of purified protein in the RNase reaction buffer (50 mM Tris-HCl at pH 8.0, 50 mM KCl, 2 mM MgCl2 , 5 mM NaH2 PO4 , and RNase IN [1 U/μL, Promega]) at 30°C for 1 h. The reaction was stopped at the time point indicated in the figures by adding TBE-Urea sample buffer (Bio-Rad) or 10 mM proteinase K. Digest patterns of 20-mer ssRNA were resolved in 20% polyacrylamide/7 M urea gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system. .. Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl.

    Purification:

    Article Title: Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3?- > 5? exonuclease containing S1 and KH RNA-binding domains
    Article Snippet: A radiolabeled in vitro transcript was made from the polylinker of plasmid pSP65(A38) ( ) linearized with Hin dIII, using SP6 RNA polymerase and [α-32 P]UTP, and gel purified. .. Ascending thin layer chromatography on polyethyleneimine–cellulose plates (Sigma) was conducted in 1 M formic acid and 0.5 M LiCl, as described previously ( ).

    Article Title: Phosphorylation of Williams Syndrome Transcription Factor by MAPK Induces a Switching between Two Distinct Chromatin Remodeling Complexes *
    Article Snippet: .. Briefly, the 5-μl reaction mixture containing 10 m m HEPES (pH 7.6), 50 m m KCl, 0.1 m m EDTA, 2 m m MgCl2 , 0.5 m m dithiothreitol, 7.5% glycerol, 0.5% Nonidet P-40, 30 μ m cold ATP, 5 μCi of [α-32 P]ATP, 20 n m plasmid DNA, and purified WINAC complexes from the stable transformant expressing FLAG-WSTF or FLAG-WSTF-S158A mutant (as described in , A and C) was incubated at 37 °C for 30 min. Hydrolyzed ATP and ADP were separated by TLC on polyethyleneimine-cellulose plates (Sigma). .. A 1-μl aliquot of the reaction mixture was spotted onto the plate, and TLC was carried out in 0.75 m KH2 PO4 .

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: For RNase activity assays, 20 nM of labeled 20-mer ssRNA or 500 ng 2.3-kb ssRNA substrates was incubated with different concentrations of purified protein in the RNase reaction buffer (50 mM Tris-HCl at pH 8.0, 50 mM KCl, 2 mM MgCl2 , 5 mM NaH2 PO4 , and RNase IN [1 U/μL, Promega]) at 30°C for 1 h. The reaction was stopped at the time point indicated in the figures by adding TBE-Urea sample buffer (Bio-Rad) or 10 mM proteinase K. Digest patterns of 20-mer ssRNA were resolved in 20% polyacrylamide/7 M urea gels, which were exposed to the phosphorimaging plate (Fujifilm) and analyzed by an FLA-5000 (Fujifilm) imaging system. .. Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl.

    Article Title: Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages *
    Article Snippet: The purified gp17s (0.2–2 μ m ), either alone or with gp16 at a gp16/gp17 molar ratio (the ratio of monomeric gp16 molecules to monomeric gp17 molecules) of 10:1, were incubated at 37 °C for 20 min in a 20-μl reaction mixture. .. EDTA was added to a final concentration of 50 m m to stop the reaction, and the ATP hydrolysis products were separated by thin layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma-Aldrich).

    Lysis:

    Article Title: The Putative "Switch 2" Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein
    Article Snippet: The beads were washed five times with lysis buffer and 32 P-labeled guanine nucleotides were eluted by incubation for 20 min at 68°C in 20 μl of elution buffer (20 mM HEPES, pH 7.3, 25 mM EDTA, 2.0% wt/vol SDS, 0.5 mM GDP, 0.5 mM GTP). .. A 5-μl aliquot of each eluate was subjected to TLC on polyethyleneimine cellulose plates (Sigma, St. Louis, MO) developed in 0.75 M KH2 PO4 (pH 3.4).

    ATPase Assay:

    Article Title: Phosphorylation of Williams Syndrome Transcription Factor by MAPK Induces a Switching between Two Distinct Chromatin Remodeling Complexes *
    Article Snippet: Paragraph title: ATPase Assay ... Briefly, the 5-μl reaction mixture containing 10 m m HEPES (pH 7.6), 50 m m KCl, 0.1 m m EDTA, 2 m m MgCl2 , 0.5 m m dithiothreitol, 7.5% glycerol, 0.5% Nonidet P-40, 30 μ m cold ATP, 5 μCi of [α-32 P]ATP, 20 n m plasmid DNA, and purified WINAC complexes from the stable transformant expressing FLAG-WSTF or FLAG-WSTF-S158A mutant (as described in , A and C) was incubated at 37 °C for 30 min. Hydrolyzed ATP and ADP were separated by TLC on polyethyleneimine-cellulose plates (Sigma).

    Plasmid Preparation:

    Article Title: Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3?- > 5? exonuclease containing S1 and KH RNA-binding domains
    Article Snippet: A radiolabeled in vitro transcript was made from the polylinker of plasmid pSP65(A38) ( ) linearized with Hin dIII, using SP6 RNA polymerase and [α-32 P]UTP, and gel purified. .. Ascending thin layer chromatography on polyethyleneimine–cellulose plates (Sigma) was conducted in 1 M formic acid and 0.5 M LiCl, as described previously ( ).

    Article Title: Phosphorylation of Williams Syndrome Transcription Factor by MAPK Induces a Switching between Two Distinct Chromatin Remodeling Complexes *
    Article Snippet: .. Briefly, the 5-μl reaction mixture containing 10 m m HEPES (pH 7.6), 50 m m KCl, 0.1 m m EDTA, 2 m m MgCl2 , 0.5 m m dithiothreitol, 7.5% glycerol, 0.5% Nonidet P-40, 30 μ m cold ATP, 5 μCi of [α-32 P]ATP, 20 n m plasmid DNA, and purified WINAC complexes from the stable transformant expressing FLAG-WSTF or FLAG-WSTF-S158A mutant (as described in , A and C) was incubated at 37 °C for 30 min. Hydrolyzed ATP and ADP were separated by TLC on polyethyleneimine-cellulose plates (Sigma). .. A 1-μl aliquot of the reaction mixture was spotted onto the plate, and TLC was carried out in 0.75 m KH2 PO4 .

    Software:

    Article Title: Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages *
    Article Snippet: EDTA was added to a final concentration of 50 m m to stop the reaction, and the ATP hydrolysis products were separated by thin layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma-Aldrich). .. The radioactive spots were quantified using ImageQuant software (GE Healthcare).

    Positron Emission Tomography:

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: The RNA substrate used in the thin layer chromatography ( ) was transcribed by T7 RNA polymerase with linearized (XhoI) pET-22b (Novagen) in the presence of [α-32 P]ATP. .. Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl.

    In Vitro:

    Article Title: Copper Ions Stimulate Polyphosphate Degradation and Phosphate Efflux in Acidithiobacillus ferrooxidans
    Article Snippet: After incubation of the mixture at 30°C for 60 min, the reaction was stopped by loading the mixture in polyethyleneimine-cellulose plates for thin-layer chromatography (Aldrich) and development in 0.75 M KH2 PO4 , pH 3.5. .. [33 P]polyP750 was synthesized in vitro by the method of Ault-Riché et al. ( ) as described in Cardona et al. ( ).

    Article Title: Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3?- > 5? exonuclease containing S1 and KH RNA-binding domains
    Article Snippet: A radiolabeled in vitro transcript was made from the polylinker of plasmid pSP65(A38) ( ) linearized with Hin dIII, using SP6 RNA polymerase and [α-32 P]UTP, and gel purified. .. Ascending thin layer chromatography on polyethyleneimine–cellulose plates (Sigma) was conducted in 1 M formic acid and 0.5 M LiCl, as described previously ( ).

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: The 2.3-kb ssRNA was transcribed in vitro using a MAXIscript kit (Ambion). .. Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl.

    Concentration Assay:

    Article Title: Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages *
    Article Snippet: .. EDTA was added to a final concentration of 50 m m to stop the reaction, and the ATP hydrolysis products were separated by thin layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma-Aldrich). .. The plates were air-dried for autoradiography and phosphorimaging (Storm 820 PhosphorImager, GE Healthcare).

    Thin Layer Chromatography:

    Article Title: Copper Ions Stimulate Polyphosphate Degradation and Phosphate Efflux in Acidithiobacillus ferrooxidans
    Article Snippet: .. After incubation of the mixture at 30°C for 60 min, the reaction was stopped by loading the mixture in polyethyleneimine-cellulose plates for thin-layer chromatography (Aldrich) and development in 0.75 M KH2 PO4 , pH 3.5. .. Radioactive spots corresponding to the Pi liberated by the hydrolysis of polyphosphate were visualized and quantified with a phosphor imager (Molecular Imager FX; Bio-Rad).

    Article Title: Analysis of Geminivirus AL2 and L2 Proteins Reveals a Novel AL2 Silencing Suppressor Activity
    Article Snippet: .. Extracts (250 ng) were incubated with adenosine and [γ-32 P]ATP (3,000 Ci/mmol; Perkin-Elmer) at 30°C for 20 min, and products were fractionated by thin-layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma) in 1 M glacial acetic acid. .. Radiolabeled AMP was quantitated using a phosphorimager (Molecular Imager FX; Bio-Rad).

    Article Title: The Putative "Switch 2" Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein
    Article Snippet: .. A 5-μl aliquot of each eluate was subjected to TLC on polyethyleneimine cellulose plates (Sigma, St. Louis, MO) developed in 0.75 M KH2 PO4 (pH 3.4). .. Radioactivity in the GTP and GDP spots was quantified with a Molecular Dynamics phosphorimaging system.

    Article Title: Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3?- > 5? exonuclease containing S1 and KH RNA-binding domains
    Article Snippet: .. Ascending thin layer chromatography on polyethyleneimine–cellulose plates (Sigma) was conducted in 1 M formic acid and 0.5 M LiCl, as described previously ( ). ..

    Article Title: Phosphorylation of Williams Syndrome Transcription Factor by MAPK Induces a Switching between Two Distinct Chromatin Remodeling Complexes *
    Article Snippet: .. Briefly, the 5-μl reaction mixture containing 10 m m HEPES (pH 7.6), 50 m m KCl, 0.1 m m EDTA, 2 m m MgCl2 , 0.5 m m dithiothreitol, 7.5% glycerol, 0.5% Nonidet P-40, 30 μ m cold ATP, 5 μCi of [α-32 P]ATP, 20 n m plasmid DNA, and purified WINAC complexes from the stable transformant expressing FLAG-WSTF or FLAG-WSTF-S158A mutant (as described in , A and C) was incubated at 37 °C for 30 min. Hydrolyzed ATP and ADP were separated by TLC on polyethyleneimine-cellulose plates (Sigma). .. A 1-μl aliquot of the reaction mixture was spotted onto the plate, and TLC was carried out in 0.75 m KH2 PO4 .

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: .. Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl. ..

    Article Title: Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages *
    Article Snippet: .. EDTA was added to a final concentration of 50 m m to stop the reaction, and the ATP hydrolysis products were separated by thin layer chromatography (TLC) on polyethyleneimine-cellulose plates (Sigma-Aldrich). .. The plates were air-dried for autoradiography and phosphorimaging (Storm 820 PhosphorImager, GE Healthcare).

    Staining:

    Article Title: Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation
    Article Snippet: For RNA-binding assays, 500 ng of 2.3-kb ssRNA substrates were incubated with different concentrations of recombinant purified protein in the RNA-binding buffer (50 mM Tris-HCl at pH 8.0, and 10 mM EDTA) on ice for 30 min. After incubation, the reaction mixture was separated on 1% agarose gels and stained with ethidium bromide. .. Ascending thin layer chromatography was done on polyethyleneimine-cellulose plates (Sigma) in 1.2 M formic acid and 0.5 M LiCl.

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    Millipore pei cellulose tlc plates
    Phosphorylation of dThd, N5 ( 3 ), N5-Br ( 12a/b ), N5-I ( 4a/b ), and N5-I 4 ( 5 ) by recombinant TK1. Assay products were separated by <t>PEI–cellulose</t> <t>TLC.</t>
    Pei Cellulose Tlc Plates, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pei cellulose tlc plates/product/Millipore
    Average 95 stars, based on 29 article reviews
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    93
    Millipore polyethyleneimine cellulose plate
    The RABV L protein generates the mRNA cap structure by an unconventional mechanism: ( a ) The recombinant RABV L protein (0.1 μg) was incubated with the indicated substrates under the standard conditions for the VSV L protein. Nuclease P 1 -digests of RNA products were analyzed by thin layer chromatography on a <t>polyethyleneimine-cellulose</t> plate (PEI-cellulose TLC), followed by autoradiography. The positions of the origin (ori.), guanosine nucleotides (GMP, GDP, GTP), and cap structures (GpppA, GppppA) are shown; ( b ) The recombinant RABV L protein (lane 2, 0.1 μg; lane 3, 0.2 μg) was incubated with [γ- 32 P]GTP. The reaction mixtures were analyzed by PEI-cellulose TLC followed by autoradiography. Lane 1 indicates no enzyme. Lane M shows the position of 32 P-labeled inorganic phosphate (P i ), which was generated by digestion of [γ- 32 P]GTP with alkaline phosphatase.
    Polyethyleneimine Cellulose Plate, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyethyleneimine cellulose plate/product/Millipore
    Average 93 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    polyethyleneimine cellulose plate - by Bioz Stars, 2020-04
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    Phosphorylation of dThd, N5 ( 3 ), N5-Br ( 12a/b ), N5-I ( 4a/b ), and N5-I 4 ( 5 ) by recombinant TK1. Assay products were separated by PEI–cellulose TLC.

    Journal: Inorganic Chemistry

    Article Title: Synthesis, biological evaluation, and radioiodination of halogenated closo-carboranyl thymidine analogues

    doi: 10.1021/ic202150b

    Figure Lengend Snippet: Phosphorylation of dThd, N5 ( 3 ), N5-Br ( 12a/b ), N5-I ( 4a/b ), and N5-I 4 ( 5 ) by recombinant TK1. Assay products were separated by PEI–cellulose TLC.

    Article Snippet: The reaction mixture was centrifuged and 2 μL sample portions were spotted on PEI–cellulose TLC plates (EMD Chemicals Inc.).

    Techniques: Recombinant, Thin Layer Chromatography

    Effect of amino acid withdrawal on [ 32 P]guanyl nucleotide content of RagC-containing heterodimers endogenous to 32 P i -labeled HEK293T cells. Replicate plates of HEK293T cells were incubated in P i -free DMEM containing 32 P i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P i -free medium ( AA +) or P i -free medium lacking amino acids ( AA -), each containing 32 P i (0.2 mCi/ml). Cells were extracted 2 h later, and immunoprecipitation ( IP ) was performed using nonimmune ( NI ) rabbit IgG or anti-RagC IgG. Nucleotides were extracted from the washed immunoprecipitates and separated by TLC on PEI cellulose. An immunoblot analysis of the extract for 4E-BP(T37P/T46P) and of the immunoprecipitates for RagC are shown in the right panels. Ori , origin.

    Journal: The Journal of Biological Chemistry

    Article Title: Amino Acids Activate Mammalian Target of Rapamycin (mTOR) Complex 1 without Changing Rag GTPase Guanyl Nucleotide Charging *

    doi: 10.1074/jbc.M113.528505

    Figure Lengend Snippet: Effect of amino acid withdrawal on [ 32 P]guanyl nucleotide content of RagC-containing heterodimers endogenous to 32 P i -labeled HEK293T cells. Replicate plates of HEK293T cells were incubated in P i -free DMEM containing 32 P i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P i -free medium ( AA +) or P i -free medium lacking amino acids ( AA -), each containing 32 P i (0.2 mCi/ml). Cells were extracted 2 h later, and immunoprecipitation ( IP ) was performed using nonimmune ( NI ) rabbit IgG or anti-RagC IgG. Nucleotides were extracted from the washed immunoprecipitates and separated by TLC on PEI cellulose. An immunoblot analysis of the extract for 4E-BP(T37P/T46P) and of the immunoprecipitates for RagC are shown in the right panels. Ori , origin.

    Article Snippet: TLC PEI cellulose F plates (catalog no. 5725-7) were from EMD Chemicals Inc.

    Techniques: Labeling, Incubation, Immunoprecipitation, Thin Layer Chromatography

    Rag heterodimers lack detectable GTPase activity in vitro . A , the RagB/C wild-type heterodimer does not hydrolyze [γ- 32 P]GTP in vitro . GST-Ha-Ras WT (▵), GST-Ha-Ras G12V (▴), and GST-RagB WT /FLAG-RagC WT (●) were charged with [γ- 32 P]GTP and examined for intrinsic GTPase activity. The amount of unhydrolysed [γ- 32 P]GTP on proteins was measured at intervals thereafter by nitrocellulose filter binding assay. B , a recombinant catalytic fragment of NF1 stimulates GTP hydrolysis by Ha-Ras WT but not Ha-Ras G12V or RagB WT /C WT . GST-Ha-Ras WT (●, dashed line ), GST-Ha-Ras G12V (○, dashed line ), or GST-RagB WT /FLAG-RagC WT (●, solid line ) were charged with [γ- 32 P]GTP, and a catalytic fragment of NF1 was added. The release of 32 P i at 30 °C was measured at intervals thereafter by the charcoal method. C , neither RagB wild-type or RagC wild-type within a heterodimer hydrolyze [α- 32 P]GTP detectably in vitro . GST and various GST-RagB/FLAG-RagC heterodimers were charged with [α- 32 P]GTP, and at intervals thereafter, nucleotides were extracted and separated on TLC on PEI cellulose. D , extracts from HEK293T cells do not promote the ability of RagB/C to hydrolyze [γ- 32 P]GTP. HEK293T cells were lysed in the absence of detergent ( solid line ) or with CHAPS (0.3%, dotted line ), TritonX-100 (1%, short dashes ), or RIPA buffer (Pierce) ( long dashes ). GST-Ha-Ras WT ( triangles ) and GST-RagB WT /FLAG-RagC WT ( circles ) charged with [γ- 32 P]GTP were mixed with each of these extracts at 30 °C, and the amount of protein-bound [γ- 32 P]GTP was measured by filtration through nitrocellulose filters. E , MonoQ anion exchange chromatography of a HEK293T cell extract contains GAP activity toward Ha-Ras wild-type, not RagB/C wild-type. HEK293T cells were extracted by freezing and thawing and separated by anion exchange chromatography. Each fraction was incubated at 30 °C with GST-Ha-Ras WT (▵, dashed lines ), GST-Ha-Ras G12V (▴, dashed lines ), and GST-RagB WT /FLAG-RagC WT , each charged with [γ- 32 P]GTP. After 30 min, protein-bound [γ- 32 P]GTP was measured as in C .

    Journal: The Journal of Biological Chemistry

    Article Title: Amino Acids Activate Mammalian Target of Rapamycin (mTOR) Complex 1 without Changing Rag GTPase Guanyl Nucleotide Charging *

    doi: 10.1074/jbc.M113.528505

    Figure Lengend Snippet: Rag heterodimers lack detectable GTPase activity in vitro . A , the RagB/C wild-type heterodimer does not hydrolyze [γ- 32 P]GTP in vitro . GST-Ha-Ras WT (▵), GST-Ha-Ras G12V (▴), and GST-RagB WT /FLAG-RagC WT (●) were charged with [γ- 32 P]GTP and examined for intrinsic GTPase activity. The amount of unhydrolysed [γ- 32 P]GTP on proteins was measured at intervals thereafter by nitrocellulose filter binding assay. B , a recombinant catalytic fragment of NF1 stimulates GTP hydrolysis by Ha-Ras WT but not Ha-Ras G12V or RagB WT /C WT . GST-Ha-Ras WT (●, dashed line ), GST-Ha-Ras G12V (○, dashed line ), or GST-RagB WT /FLAG-RagC WT (●, solid line ) were charged with [γ- 32 P]GTP, and a catalytic fragment of NF1 was added. The release of 32 P i at 30 °C was measured at intervals thereafter by the charcoal method. C , neither RagB wild-type or RagC wild-type within a heterodimer hydrolyze [α- 32 P]GTP detectably in vitro . GST and various GST-RagB/FLAG-RagC heterodimers were charged with [α- 32 P]GTP, and at intervals thereafter, nucleotides were extracted and separated on TLC on PEI cellulose. D , extracts from HEK293T cells do not promote the ability of RagB/C to hydrolyze [γ- 32 P]GTP. HEK293T cells were lysed in the absence of detergent ( solid line ) or with CHAPS (0.3%, dotted line ), TritonX-100 (1%, short dashes ), or RIPA buffer (Pierce) ( long dashes ). GST-Ha-Ras WT ( triangles ) and GST-RagB WT /FLAG-RagC WT ( circles ) charged with [γ- 32 P]GTP were mixed with each of these extracts at 30 °C, and the amount of protein-bound [γ- 32 P]GTP was measured by filtration through nitrocellulose filters. E , MonoQ anion exchange chromatography of a HEK293T cell extract contains GAP activity toward Ha-Ras wild-type, not RagB/C wild-type. HEK293T cells were extracted by freezing and thawing and separated by anion exchange chromatography. Each fraction was incubated at 30 °C with GST-Ha-Ras WT (▵, dashed lines ), GST-Ha-Ras G12V (▴, dashed lines ), and GST-RagB WT /FLAG-RagC WT , each charged with [γ- 32 P]GTP. After 30 min, protein-bound [γ- 32 P]GTP was measured as in C .

    Article Snippet: TLC PEI cellulose F plates (catalog no. 5725-7) were from EMD Chemicals Inc.

    Techniques: Activity Assay, In Vitro, Filter-binding Assay, Recombinant, Thin Layer Chromatography, Filtration, Chromatography, Incubation

    The effect of amino acid withdrawal and insulin on the [ 32 P]guanyl nucleotide content of stably expressed RagC WT and RagC S75L heterodimeric complexes in 32 P i -labeled HeLa cells. A , B , and C , replicate plates of HeLa cells stably expressing GFP-streptag, GFP-streptag-RagC WT , or GFP-streptag-RagC S75L were incubated in P i -free DMEM containing 32 P i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P i -free medium ( AA +) or P i -free medium lacking amino acids ( AA -), each containing 32 P i (0.2 mCi/ml), for another 2 h. In A and B , insulin (1.0 μ m ) was added to some of the cells in DMEM ( AA +/ I ) 30 min before harvest. The nucleotides bound to Strep-Tactin ( strept ) pull-downs were extracted and separated by TLC on PEI cellulose. One set of 32 P -labeled HeLa cells expressing FP-streptag, treated as described above for AA-, AA+, and AA+/I, were rinsed, extracted directly into acetonitrile, and then the solubilized total nucleotides were separated by TLC. The 32 P comigrating with GDP and GTP was quantitated by phosphorimaging. After subtraction of the averaged values found in the GFP-streptag lanes, the percentage of [ 32 P]GTP was calculated as [[ 32 P]GTP / (1.5 × [ 32 P]GDP + [ 32 P]GTP)], and the averaged values are shown. In A , the top and center panels on the right show GFP immunoblot analyses of the cell lysates and representative Strep-Tactin pull-downs (corresponding to lanes 1 , 4 , and 7 of each set). The bottom panel shows an immunoblot analysis of S6K(T389P) corresponding to lanes 1 , 4 , and 7 of each set. In B , the top panel on the right shows a GFP immunoblot analysis of the lysates, whereas the center and bottom panels show lysate immunoblot analyses for 4E-BP(T37P/T46P) and PRAS40(S246P), respectively. In C , one set of 32 P-labeled HeLa cells expressing GFP-streptag, treated as in Fig. 6 , were rinsed, extracted directly into HClO 4 (0.3 m , 0 °C, HClO 4 extract ). The HClO 4 supernatants were neutralized with KHCO 3 , and the [ 32 P]guanyl nucleotides were quantified as above. In addition, the nucleotides in the lysate after pull-down of the Strep-Tactin beads were also analyzed. Immunoblot analyses of the lysates and representative Strep-Tactin pull-downs are shown in the right panel. Ori , origin; std , guanyl nucleotide standards; Ins , insulin; ACN , acetonitrile; Ct , C-terminal.

    Journal: The Journal of Biological Chemistry

    Article Title: Amino Acids Activate Mammalian Target of Rapamycin (mTOR) Complex 1 without Changing Rag GTPase Guanyl Nucleotide Charging *

    doi: 10.1074/jbc.M113.528505

    Figure Lengend Snippet: The effect of amino acid withdrawal and insulin on the [ 32 P]guanyl nucleotide content of stably expressed RagC WT and RagC S75L heterodimeric complexes in 32 P i -labeled HeLa cells. A , B , and C , replicate plates of HeLa cells stably expressing GFP-streptag, GFP-streptag-RagC WT , or GFP-streptag-RagC S75L were incubated in P i -free DMEM containing 32 P i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P i -free medium ( AA +) or P i -free medium lacking amino acids ( AA -), each containing 32 P i (0.2 mCi/ml), for another 2 h. In A and B , insulin (1.0 μ m ) was added to some of the cells in DMEM ( AA +/ I ) 30 min before harvest. The nucleotides bound to Strep-Tactin ( strept ) pull-downs were extracted and separated by TLC on PEI cellulose. One set of 32 P -labeled HeLa cells expressing FP-streptag, treated as described above for AA-, AA+, and AA+/I, were rinsed, extracted directly into acetonitrile, and then the solubilized total nucleotides were separated by TLC. The 32 P comigrating with GDP and GTP was quantitated by phosphorimaging. After subtraction of the averaged values found in the GFP-streptag lanes, the percentage of [ 32 P]GTP was calculated as [[ 32 P]GTP / (1.5 × [ 32 P]GDP + [ 32 P]GTP)], and the averaged values are shown. In A , the top and center panels on the right show GFP immunoblot analyses of the cell lysates and representative Strep-Tactin pull-downs (corresponding to lanes 1 , 4 , and 7 of each set). The bottom panel shows an immunoblot analysis of S6K(T389P) corresponding to lanes 1 , 4 , and 7 of each set. In B , the top panel on the right shows a GFP immunoblot analysis of the lysates, whereas the center and bottom panels show lysate immunoblot analyses for 4E-BP(T37P/T46P) and PRAS40(S246P), respectively. In C , one set of 32 P-labeled HeLa cells expressing GFP-streptag, treated as in Fig. 6 , were rinsed, extracted directly into HClO 4 (0.3 m , 0 °C, HClO 4 extract ). The HClO 4 supernatants were neutralized with KHCO 3 , and the [ 32 P]guanyl nucleotides were quantified as above. In addition, the nucleotides in the lysate after pull-down of the Strep-Tactin beads were also analyzed. Immunoblot analyses of the lysates and representative Strep-Tactin pull-downs are shown in the right panel. Ori , origin; std , guanyl nucleotide standards; Ins , insulin; ACN , acetonitrile; Ct , C-terminal.

    Article Snippet: TLC PEI cellulose F plates (catalog no. 5725-7) were from EMD Chemicals Inc.

    Techniques: Stable Transfection, Labeling, Expressing, Incubation, Thin Layer Chromatography

    Phosphorylation rates of dThd, N5-2OH and N3-substituted dThd analogues IPwith hTK1. Assay products were separated by PEI–cellulose TLC and quantified by β -radiography. ( A ). Compounds 3e, 3f and 7e-7g ; (a) dThd monophosphate (dTMP), (b)

    Journal: European journal of medicinal chemistry

    Article Title: Synthesis of N3-Substituted Carboranyl Thymidine Bioconjugates and their Evaluation as Substrates of Recombinant Human Thymidine Kinase 1

    doi: 10.1016/j.ejmech.2012.11.041

    Figure Lengend Snippet: Phosphorylation rates of dThd, N5-2OH and N3-substituted dThd analogues IPwith hTK1. Assay products were separated by PEI–cellulose TLC and quantified by β -radiography. ( A ). Compounds 3e, 3f and 7e-7g ; (a) dThd monophosphate (dTMP), (b)

    Article Snippet: The reaction mixtures were centrifuged and 2 μL sample portions were spotted on PEI–cellulose TLC plates (EMD Chemicals Inc.).

    Techniques: Thin Layer Chromatography

    The RABV L protein generates the mRNA cap structure by an unconventional mechanism: ( a ) The recombinant RABV L protein (0.1 μg) was incubated with the indicated substrates under the standard conditions for the VSV L protein. Nuclease P 1 -digests of RNA products were analyzed by thin layer chromatography on a polyethyleneimine-cellulose plate (PEI-cellulose TLC), followed by autoradiography. The positions of the origin (ori.), guanosine nucleotides (GMP, GDP, GTP), and cap structures (GpppA, GppppA) are shown; ( b ) The recombinant RABV L protein (lane 2, 0.1 μg; lane 3, 0.2 μg) was incubated with [γ- 32 P]GTP. The reaction mixtures were analyzed by PEI-cellulose TLC followed by autoradiography. Lane 1 indicates no enzyme. Lane M shows the position of 32 P-labeled inorganic phosphate (P i ), which was generated by digestion of [γ- 32 P]GTP with alkaline phosphatase.

    Journal: Viruses

    Article Title: The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity

    doi: 10.3390/v8050144

    Figure Lengend Snippet: The RABV L protein generates the mRNA cap structure by an unconventional mechanism: ( a ) The recombinant RABV L protein (0.1 μg) was incubated with the indicated substrates under the standard conditions for the VSV L protein. Nuclease P 1 -digests of RNA products were analyzed by thin layer chromatography on a polyethyleneimine-cellulose plate (PEI-cellulose TLC), followed by autoradiography. The positions of the origin (ori.), guanosine nucleotides (GMP, GDP, GTP), and cap structures (GpppA, GppppA) are shown; ( b ) The recombinant RABV L protein (lane 2, 0.1 μg; lane 3, 0.2 μg) was incubated with [γ- 32 P]GTP. The reaction mixtures were analyzed by PEI-cellulose TLC followed by autoradiography. Lane 1 indicates no enzyme. Lane M shows the position of 32 P-labeled inorganic phosphate (P i ), which was generated by digestion of [γ- 32 P]GTP with alkaline phosphatase.

    Article Snippet: Calf intestine alkaline phosphatase and nuclease P1 -resistant products were analyzed together with standard nucleotides (GMP, GDP, GTP, G(5′)ppp(5′)A (New England Biolabs, Ipswich, MA, USA), and/or G(5′)ppp(5′)G (New England Biolabs)) by thin layer chromatography on a polyethyleneimine-cellulose plate (EMD Millipore, Billerica, MA, USA) (PEI-cellulose TLC) as described [ ].

    Techniques: Recombinant, Incubation, Thin Layer Chromatography, Autoradiography, Labeling, Generated