s l methionine  (PerkinElmer)

 
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    Name:
    Adenosyl L Methionine S methyl 14C 10µCi 370kBq
    Description:
    10 µCi quantities of S methyl 14C Adenosyl L Methionine are available for your research Application of 14C SAM can be found in methylation in epigenetics a new caffeine biosynthetic pathway in tea leaves in phytochemistry effect of salt stress on the metabolism of ethanolamine and choline in mangrove leaves in phytochemisty pharmacokinetic of the orally administered salt in rats etc Need more information
    Catalog Number:
    NEC363010UC
    Price:
    None
    Size:
    10 µCi
    Category:
    Reagents
    Score:
    85
    Buy from Supplier
    Name:
    Adenosyl L Methionine S methyl 3H Specific Activity 55 85Ci 2 03 3 15TBq mMole 250µCi 9 25MBq
    Description:
    Tritiated SAM is commonly used in epigenetic methylation assays
    Catalog Number:
    NET155H250UC
    Price:
    None
    Size:
    250 µCi
    Category:
    Reagents
    Score:
    85
    Buy from Supplier


    Structured Review

    PerkinElmer s l methionine
    Adenosyl L Methionine S methyl 3H Specific Activity 55 85Ci 2 03 3 15TBq mMole 250µCi 9 25MBq
    Tritiated SAM is commonly used in epigenetic methylation assays
    https://www.bioz.com/result/s l methionine/product/PerkinElmer
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    s l methionine - by Bioz Stars, 2019-10
    89/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries
    Article Snippet: Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]). .. After labeling, cells were washed twice with excess phosphate-buffered saline (PBS) before lysis with cold radioimmunoprecipitation assay (RIPA) buffer supplemented with complete protease inhibitor cocktail (Sigma-Aldrich).

    Synthesized:

    Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
    Article Snippet: His-tagged MetRS was purified from Escherichia coli XL1 Blue cells (Agilent) carrying the E. coli methionyl-tRNA synthetase (MetRS) expression plasmid pRA101 ( ) as described previously ( ). .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ). .. For measurements of tRNA binding, seven 2-fold serial dilutions of eIF2 with final concentrations from 3.125 to 200 nM were incubated for 15 min at 26°C in Binding Assay Buffer (25 mM HEPES-KOH (pH 7.6), 2.5 mM Mg(OAc)2 , 80 mM KOAc (pH 7.6), 2 mM DTT and 0.5 mM guanosine 5′-[β,γ-imido]triphosphate (GDPNP)-Mg2+ ).

    Article Title: The Rab6-regulated KIF1C kinesin motor domain contributes to Golgi organization
    Article Snippet: The eluate was diluted fivefold with Buffer C (30 mM Hepes, pH 7.4, 1 mM MgCl2 , 1 mM EGTA, 25 µM ATP) and applied to HiTrap Q FF (GE Healthcare), washed with Buffer C + 100 mM NaCl, before being eluted by gradient to 500 mM NaCl in Buffer C. Binding of the purified components was assayed in the same manner as in vitro translation-synthesized constructs. .. Full-length myc-KIF1C synthesized in vitro with 35 S-methionine (EasyTag, Perkin Elmer, San Jose, CA) was desalted and incubated with GTPγS-preloaded Rabs (4.2 μM) for 1 hr in Buffer A (−BSA), 1 mM DTT, 2.5 mM ADP, 0.5 mM GTPγS. .. Microtubules (0.8 mg/ml), polymerized in 80 mM PIPES, 1 mM MgCl2 , 1 mM EGTA, pH 6.8 (BRB80), 1 mM DTT, 1 mM GTP, 10% DMSO, spun through a 40% glycerol cushion, and resuspended in BRB80, 1 mM DTT, 0.2 mM Paclitaxel (Cytoskeleton, Inc.), were incubated with the KIF1C-Rab complexes for 1 hr before being spun through a 10% sucrose, 20 µM Paclitaxel, 1 mM DTT, at 65K for 5 min (Optima TLX, Beckman Coulter, Inc., Indianapolis, IN).

    Autoradiography:

    Article Title: Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants
    Article Snippet: CEM-T4 T cells were starved for 20 min in methionine-free, cysteine-free RPMI/5% dialysed FCS (Invitrogen), labeled with [35 S]methionine/[35 S]cysteine (EasyTag EXPRESS, PerkinElmer) for 15 min, then chased in RPMI/10% FCS at 37°C. .. Cells were lysed in 1% Triton X-100 at the indicated timepoints, and subjected to immunoprecipitation with anti-PPP2R5D as described.

    Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
    Article Snippet: Ubiquitination reactions were carried out with a total volume of 20 μl containing 2 μg of ubiquitin, 20 pmol of E1, and 100 pmol of E2 as well as 2 mM ATP at 37°C for 60 min. Boiling in SDS-sample buffer terminated the reactions, and the products were analyzed by SDS-PAGE and autoradiography. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

    Article Title: Adult-onset obesity is triggered by impaired mitochondrial gene expression
    Article Snippet: Mitochondria were supplemented with 150 μCi of [35 S]methionine (PerkinElmer) for 60 min at 37°C. .. After labeling, mitochondria were washed in translation buffer and suspended in radioimmunoprecipitation assay lysis buffer.

    Blocking Assay:

    Article Title: Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries
    Article Snippet: Twenty fractions (100 µl each) were collected, and 10-µl aliquots of the first 17 fractions containing mitoribosomal subunits and assembled mitoribosomes were analyzed by WB. .. Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]). .. For total cellular translation, 25 µCi/ml was used without emetine.

    Ubiquitin Assay:

    Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
    Article Snippet: For the in vitro ubiquitination assay, GST-PRLR proteins were expressed in bacteria, purified by using glutathione beads, and preincubated (1 μg) with 25-μg extracts from 293T cells treated with PRL (200 ng/ml for 20 min) in a kinase buffer containing 50 mM Tris HCl (pH 7.5), 5 mM MgCl2 , 0.5 mM dithiothreitol, 5 mM NaF, 10 nM okadaic acid, 50 μM ATP, and 10 μM [32 P-γ]ATP for 30 min at 30°C. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

    Incubation:

    Article Title: Altered intracellular calcium homeostasis and endoplasmic reticulum redox state in Saccharomyces cerevisiae cells lacking Grx6 glutaredoxin
    Article Snippet: Background values were determined for a region lacking visible signal of the same size as the measured band and adjacent to it, and such background was subtracted for the respective band signal value. .. Samples (∼6 × 108 cells) from cultures in SC medium were resuspended in 10 ml of the same medium lacking methionine and cysteine and incubated for 30 min at the appropriate temperature before adding 500 μCi of a [35 S]methionine/[35 S]cysteine cocktail (Perkin Elmer, Waltham, MA). .. After a 5- or 6-min pulse, cold methionine and cysteine were added at 2 mM final concentration each.

    Article Title: Mitochondrial translation requires folate-dependent tRNA methylation
    Article Snippet: Then media was changed to DMEM with 10 % dFBS without methionine (MP Biomedicals). .. Following a 30 min incubation, cytosolic translation was inhibited by emetine hydrochloride (0.1 mg/ml, Sigma-Aldrich) and labeling was conducted for one hour after the addition of 500 μCi [35 S]-methionine (EasyTag™ L[ S]-Methionine, Perkin Elmer). .. Protein lysates (30 μg) were then separated on a 15% polyacrylamide gel (8.3 × 7.3 cm) and dried using a 443 Slab Dryer (BioRad).

    Article Title: The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis
    Article Snippet: Briefly WT and Top1mt -KO MEFs were washed with methionine/cysteine-free DMEM, 2 mM l -glutamine, 96 μg/mL cysteine and 5% (v/v) dialyzed FBS and incubated for 10 min in the same media at 37 °C. .. 100 μg/mL emetine was then added directly to the media in order to inhibit cytosolic translation; after 20 min proteins newly synthetized in the mitochondrial compartment were labeled with 100 μCi [35 S]-methionine (Perkin Elmer) for 1 h at 37 °C.

    Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
    Article Snippet: His-tagged MetRS was purified from Escherichia coli XL1 Blue cells (Agilent) carrying the E. coli methionyl-tRNA synthetase (MetRS) expression plasmid pRA101 ( ) as described previously ( ). .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ). .. For measurements of tRNA binding, seven 2-fold serial dilutions of eIF2 with final concentrations from 3.125 to 200 nM were incubated for 15 min at 26°C in Binding Assay Buffer (25 mM HEPES-KOH (pH 7.6), 2.5 mM Mg(OAc)2 , 80 mM KOAc (pH 7.6), 2 mM DTT and 0.5 mM guanosine 5′-[β,γ-imido]triphosphate (GDPNP)-Mg2+ ).

    Article Title: Reduced phosphorylation of ribosomal protein S6 is associated with sensitivity to MEK inhibition in gastric cancer cells
    Article Snippet: Finally, the signals were detected using an ECL Western blotting analysis system (GE Healthcare, Piscataway, NJ, USA) in accordance with the manufacturer's instructions. .. To determine the rate of protein synthesis, cells treated with DMSO or 1 μM PD0325901 were incubated with methionine‐free RPMI‐1640 for 10 min, and then labeled with 2 MBq/mL [35 S]methionine (PerkinElmer Japan, Tokyo, Japan) for 20 min. After a wash with PBS, the cells were lysed with a Total Protein Extraction Kit (BioChain, Hayward, CA, USA) in accordance with the manufacturer's instructions and centrifuged at 17 400 g for 20 min. .. The resulting cell lysates (40 μg/sample) were resuspended in Laemmli sample buffer and subjected to SDS‐PAGE using 10% gel.

    Article Title: In vitro centromere and kinetochore assembly on defined chromatin templates
    Article Snippet: Human and Xenopus CENP-C was in vitro translated (IVT) in rabbit reticulocyte extracts in the presence of 10 mCi/Ml [35 S]methionine (Perkin Elmer) using the TnT Quick-Coupled Transcription/Translation system (Promega) according to the manufacturer’s instructions. .. For a binding reaction (60μl total volume), 5 μl of each IVT protein were mixed with chromatin arrays in bead buffer (75mM Tris-HCl pH 7.5, 50mM NaCl, 0.25mM EDTA, 0.05% Triton-X-100).

    Article Title: Adult-onset obesity is triggered by impaired mitochondrial gene expression
    Article Snippet: Briefly, 500 μg of mitochondria was incubated in 750 μl of translation buffer [100 mM mannitol, 10 mM sodium succinate, 80 mM KCl, 5 mM MgCl2 , 1 mM KPi, 25 mM Hepes (pH 7.4), 5 mM ATP, 20 μM guanosine triphosphate, 6 mM creatine phosphate, creatine kinase (60 μg/ml), and 60 μg/ml of all amino acids except methionine]. .. Mitochondria were supplemented with 150 μCi of [35 S]methionine (PerkinElmer) for 60 min at 37°C.

    Article Title: Downregulation of ribosome biogenesis during early forebrain development
    Article Snippet: E8.5 and E10.5 forebrain neuroepithelium was dissected as described ( ) and trypsinized. .. Cells were serum starved in methionine-free DMEM for 1 hr at 37°C, then incubated with 51 μCi 35 S-Methionine (Perkin Elmer NEG709A) at 37°C for an additional hour. .. Cycloheximide (50 μg/ml) was added to stop translation.

    Article Title: Non-canonical translation initiation of the spliced mRNA encoding the human T-cell leukemia virus type 1 basic leucine zipper protein
    Article Snippet: Other monocistronic mRNAs were translated in RRL standard salt conditions (40 mM KOAc and 0.25 mM MgOAc2 ). .. All translation reaction mixtures were incubated at 30°C for 90 min. For 35 S-methionine labeling, the RRL (Flexi® -RRL; #L4540; Promega) reactions were performed as described in , using 0.02 mM of amino acids lacking methionine and supplemented with 0.6 mCi/ ml [35 S]-methionine (PerkinElmer, Inc., Waltham, MA, USA). .. The translation reaction was stopped with 90 μl of protein loading buffer (10% sodium dodecyl sulphate, 50 mM Tris (pH 6.8), 10% glycerol, 100 mM DTT and 0.1% bromophenol blue) and 10 μl of the final reaction was resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (12%), and the labeled products were visualized and quantified using a Storm PhosphorImager (GE Healthcare) as specified in ( ).

    Article Title: The Rab6-regulated KIF1C kinesin motor domain contributes to Golgi organization
    Article Snippet: The eluate was diluted fivefold with Buffer C (30 mM Hepes, pH 7.4, 1 mM MgCl2 , 1 mM EGTA, 25 µM ATP) and applied to HiTrap Q FF (GE Healthcare), washed with Buffer C + 100 mM NaCl, before being eluted by gradient to 500 mM NaCl in Buffer C. Binding of the purified components was assayed in the same manner as in vitro translation-synthesized constructs. .. Full-length myc-KIF1C synthesized in vitro with 35 S-methionine (EasyTag, Perkin Elmer, San Jose, CA) was desalted and incubated with GTPγS-preloaded Rabs (4.2 μM) for 1 hr in Buffer A (−BSA), 1 mM DTT, 2.5 mM ADP, 0.5 mM GTPγS. .. Microtubules (0.8 mg/ml), polymerized in 80 mM PIPES, 1 mM MgCl2 , 1 mM EGTA, pH 6.8 (BRB80), 1 mM DTT, 1 mM GTP, 10% DMSO, spun through a 40% glycerol cushion, and resuspended in BRB80, 1 mM DTT, 0.2 mM Paclitaxel (Cytoskeleton, Inc.), were incubated with the KIF1C-Rab complexes for 1 hr before being spun through a 10% sucrose, 20 µM Paclitaxel, 1 mM DTT, at 65K for 5 min (Optima TLX, Beckman Coulter, Inc., Indianapolis, IN).

    Article Title: Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA
    Article Snippet: After incubation, 25 μM of MG132 proteasome inhibitor was added to the medium and cells were incubated for 45 min. .. Cells were then cultured in a medium containing 0.15 mCi ml–1 35 S-methionine (Perkin Elmer, Boston, USA) for 1 h and collected.

    Article Title: Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries
    Article Snippet: Twenty fractions (100 µl each) were collected, and 10-µl aliquots of the first 17 fractions containing mitoribosomal subunits and assembled mitoribosomes were analyzed by WB. .. Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]). .. For total cellular translation, 25 µCi/ml was used without emetine.

    Article Title: Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis
    Article Snippet: For aminoacylation of yeast initiator tRNAi Met , E. coli MetRS was purified from E. coli strain XL1 Blue (Agilent) transformed with pRA101 ( ) and yeast tRNAi Met was prepared by in vitro transcription as described previously ( ). .. Aminoacylation reactions containing 5 μM tRNAi Met , 2 mM ATP, 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS in 1× reaction buffer A (100 mM HEPES–KOH [pH 7.5], 10 mM KCl and 1 mM DTT) were incubated at 37°C for 30 min. .. In typical reactions ∼50–60% of tRNAi Met was charged with [35 S]methionine.

    Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
    Article Snippet: Radiolabeled P1-2A was generated from plasmids using in vitro transcription and translation reactions in rabbit reticulocyte lysates (TnT quick; Promega) according to the manufacturer's instructions. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS. .. Products of processing reactions were separated by SDS-PAGE.

    Article Title: Paired Ig-Like Type 2 Receptor-Derived Agonist Ligands Ameliorate Inflammatory Reactions by Downregulating β1 Integrin Activity
    Article Snippet: Mouse WEHI274.1 monocytes were suspended and incubated in methionine-free medium to deplete intracellular methionine for 15 min at 37°C under 5% CO2 . .. Labeling medium containing 0.2 mCi/ml of [35 S]-methionine (PerkinElmer Inc., USA) was added, followed by incubation for 30 min at 37°C. .. Methionine-containing medium was added into the cell suspension.

    Expressing:

    Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
    Article Snippet: His-tagged MetRS was purified from Escherichia coli XL1 Blue cells (Agilent) carrying the E. coli methionyl-tRNA synthetase (MetRS) expression plasmid pRA101 ( ) as described previously ( ). .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ).

    Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
    Article Snippet: A plasmid encoding the capsid and 3C protease genome regions of the O1 Manisa strain of FMDV in pBG200 T7 expression vectors ( ) were used as a template to generate the plasmids pBG200-P1-2A and pBG200-P1-Δ2A, with the primer sequences described in , by PCR. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

    Transformation Assay:

    Article Title: Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis
    Article Snippet: For aminoacylation of yeast initiator tRNAi Met , E. coli MetRS was purified from E. coli strain XL1 Blue (Agilent) transformed with pRA101 ( ) and yeast tRNAi Met was prepared by in vitro transcription as described previously ( ). .. Aminoacylation reactions containing 5 μM tRNAi Met , 2 mM ATP, 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS in 1× reaction buffer A (100 mM HEPES–KOH [pH 7.5], 10 mM KCl and 1 mM DTT) were incubated at 37°C for 30 min.

    Transfection:

    Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
    Article Snippet: Briefly, cells were grown in 100-mm-diameter dishes and transfected with the indicated plasmids. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

    Article Title: Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA
    Article Snippet: Forty hours after the transfection, cells were incubated 30 min in a methionine-free medium. .. Cells were then cultured in a medium containing 0.15 mCi ml–1 35 S-methionine (Perkin Elmer, Boston, USA) for 1 h and collected.

    Pulse Chase:

    Article Title: Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants
    Article Snippet: Paragraph title: Pulse-chase ... CEM-T4 T cells were starved for 20 min in methionine-free, cysteine-free RPMI/5% dialysed FCS (Invitrogen), labeled with [35 S]methionine/[35 S]cysteine (EasyTag EXPRESS, PerkinElmer) for 15 min, then chased in RPMI/10% FCS at 37°C.

    Article Title: Altered intracellular calcium homeostasis and endoplasmic reticulum redox state in Saccharomyces cerevisiae cells lacking Grx6 glutaredoxin
    Article Snippet: Paragraph title: Pulse-chase labeling and immunoprecipitation of CPY ... Samples (∼6 × 108 cells) from cultures in SC medium were resuspended in 10 ml of the same medium lacking methionine and cysteine and incubated for 30 min at the appropriate temperature before adding 500 μCi of a [35 S]methionine/[35 S]cysteine cocktail (Perkin Elmer, Waltham, MA).

    Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
    Article Snippet: Pulse-chase analysis was carried out with 293T cells as described elsewhere ( ). .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

    Concentration Assay:

    Article Title: Altered intracellular calcium homeostasis and endoplasmic reticulum redox state in Saccharomyces cerevisiae cells lacking Grx6 glutaredoxin
    Article Snippet: Samples (∼6 × 108 cells) from cultures in SC medium were resuspended in 10 ml of the same medium lacking methionine and cysteine and incubated for 30 min at the appropriate temperature before adding 500 μCi of a [35 S]methionine/[35 S]cysteine cocktail (Perkin Elmer, Waltham, MA). .. Samples (∼6 × 108 cells) from cultures in SC medium were resuspended in 10 ml of the same medium lacking methionine and cysteine and incubated for 30 min at the appropriate temperature before adding 500 μCi of a [35 S]methionine/[35 S]cysteine cocktail (Perkin Elmer, Waltham, MA).

    Protease Inhibitor:

    Article Title: Altered intracellular calcium homeostasis and endoplasmic reticulum redox state in Saccharomyces cerevisiae cells lacking Grx6 glutaredoxin
    Article Snippet: Samples (∼6 × 108 cells) from cultures in SC medium were resuspended in 10 ml of the same medium lacking methionine and cysteine and incubated for 30 min at the appropriate temperature before adding 500 μCi of a [35 S]methionine/[35 S]cysteine cocktail (Perkin Elmer, Waltham, MA). .. Samples (∼6 × 108 cells) from cultures in SC medium were resuspended in 10 ml of the same medium lacking methionine and cysteine and incubated for 30 min at the appropriate temperature before adding 500 μCi of a [35 S]methionine/[35 S]cysteine cocktail (Perkin Elmer, Waltham, MA).

    Article Title: Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries
    Article Snippet: Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]). .. Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]).

    Cell Culture:

    Article Title: Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA
    Article Snippet: After incubation, 25 μM of MG132 proteasome inhibitor was added to the medium and cells were incubated for 45 min. .. Cells were then cultured in a medium containing 0.15 mCi ml–1 35 S-methionine (Perkin Elmer, Boston, USA) for 1 h and collected. .. Cell pellets were suspended in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Igepal and treated as described above.

    Hemagglutination Assay:

    Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
    Article Snippet: Proteins were captured on SCFβ-TrCP isolated from transfected 293T cells with HA antibody (Roche) and protein A beads (Invitrogen) as previously described ( ) and washed with buffer containing 300 mM NaCl, 0.5% Nonidet P-40, and phosphatase inhibitors. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

    Generated:

    Article Title: Non-canonical translation initiation of the spliced mRNA encoding the human T-cell leukemia virus type 1 basic leucine zipper protein
    Article Snippet: When HeLa cell extracts (S10) were used, the dl HIV-1 IRES bicistronic mRNA was preincubated for 10 min with 0.1 μg of cell extracts generated from G2/M arrested cells prior to the addition of the RRL translation mix as described previously ( , ). .. All translation reaction mixtures were incubated at 30°C for 90 min. For 35 S-methionine labeling, the RRL (Flexi® -RRL; #L4540; Promega) reactions were performed as described in , using 0.02 mM of amino acids lacking methionine and supplemented with 0.6 mCi/ ml [35 S]-methionine (PerkinElmer, Inc., Waltham, MA, USA).

    Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
    Article Snippet: Radiolabeled P1-2A was generated from plasmids using in vitro transcription and translation reactions in rabbit reticulocyte lysates (TnT quick; Promega) according to the manufacturer's instructions. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

    Protein Concentration:

    Article Title: Adult-onset obesity is triggered by impaired mitochondrial gene expression
    Article Snippet: Mitochondria were supplemented with 150 μCi of [35 S]methionine (PerkinElmer) for 60 min at 37°C. .. After labeling, mitochondria were washed in translation buffer and suspended in radioimmunoprecipitation assay lysis buffer.

    Sequencing:

    Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
    Article Snippet: PCRs were performed using KOD polymerase (Roche), and a VP0 G2A substitution to prevent myristoylation was introduced into the coding sequence of pBG200-P1-Δ2A to generate pBG200-P1-2A-G2A using QuikChange lightning mutagenesis (Agilent) and the primer sequences described in . .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

    Binding Assay:

    Article Title: In vitro centromere and kinetochore assembly on defined chromatin templates
    Article Snippet: Paragraph title: In vitro binding of centromere proteins to chromatin arrays ... Human and Xenopus CENP-C was in vitro translated (IVT) in rabbit reticulocyte extracts in the presence of 10 mCi/Ml [35 S]methionine (Perkin Elmer) using the TnT Quick-Coupled Transcription/Translation system (Promega) according to the manufacturer’s instructions.

    Article Title: The Rab6-regulated KIF1C kinesin motor domain contributes to Golgi organization
    Article Snippet: Paragraph title: Radioactive KIF1C Microtubule binding ... Full-length myc-KIF1C synthesized in vitro with 35 S-methionine (EasyTag, Perkin Elmer, San Jose, CA) was desalted and incubated with GTPγS-preloaded Rabs (4.2 μM) for 1 hr in Buffer A (−BSA), 1 mM DTT, 2.5 mM ADP, 0.5 mM GTPγS.

    Transmission Electron Microscopy:

    Article Title: Paired Ig-Like Type 2 Receptor-Derived Agonist Ligands Ameliorate Inflammatory Reactions by Downregulating β1 Integrin Activity
    Article Snippet: Paragraph title: In vivo TEM assay ... Labeling medium containing 0.2 mCi/ml of [35 S]-methionine (PerkinElmer Inc., USA) was added, followed by incubation for 30 min at 37°C.

    Nucleic Acid Electrophoresis:

    Article Title: Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication
    Article Snippet: Pulse-labeling of HepG2 cells with [35 S]methionine was performed as previously described ( ). .. Cells were treated with drugs as described above for 24 h before they were washed with media lacking methionine for 1 h. Cultures were then pulsed with [35 S]methionine (200 µCi/well EasyTag express protein labeling mix; PerkinElmer) for 45 min, lysed, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). .. The gel was dried and exposed to phosphor screen for 24 h before being imaged on a Bio-Rad personal molecular imager system.

    In Vivo:

    Article Title: Paired Ig-Like Type 2 Receptor-Derived Agonist Ligands Ameliorate Inflammatory Reactions by Downregulating β1 Integrin Activity
    Article Snippet: Paragraph title: In vivo TEM assay ... Labeling medium containing 0.2 mCi/ml of [35 S]-methionine (PerkinElmer Inc., USA) was added, followed by incubation for 30 min at 37°C.

    Radioactivity:

    Article Title: Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication
    Article Snippet: Paragraph title: [35 S]methionine radioactivity assay. ... Cells were treated with drugs as described above for 24 h before they were washed with media lacking methionine for 1 h. Cultures were then pulsed with [35 S]methionine (200 µCi/well EasyTag express protein labeling mix; PerkinElmer) for 45 min, lysed, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

    Magnetic Beads:

    Article Title: Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA
    Article Snippet: Cells were then cultured in a medium containing 0.15 mCi ml–1 35 S-methionine (Perkin Elmer, Boston, USA) for 1 h and collected. .. Cells were then cultured in a medium containing 0.15 mCi ml–1 35 S-methionine (Perkin Elmer, Boston, USA) for 1 h and collected.

    Mutagenesis:

    Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
    Article Snippet: PCRs were performed using KOD polymerase (Roche), and a VP0 G2A substitution to prevent myristoylation was introduced into the coding sequence of pBG200-P1-Δ2A to generate pBG200-P1-2A-G2A using QuikChange lightning mutagenesis (Agilent) and the primer sequences described in . .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

    Isolation:

    Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
    Article Snippet: Proteins were captured on SCFβ-TrCP isolated from transfected 293T cells with HA antibody (Roche) and protein A beads (Invitrogen) as previously described ( ) and washed with buffer containing 300 mM NaCl, 0.5% Nonidet P-40, and phosphatase inhibitors. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

    Article Title: Adult-onset obesity is triggered by impaired mitochondrial gene expression
    Article Snippet: In organello translation assays were carried out in isolated heart and liver mitochondria, as described previously ( ). .. Mitochondria were supplemented with 150 μCi of [35 S]methionine (PerkinElmer) for 60 min at 37°C.

    Labeling:

    Article Title: Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants
    Article Snippet: Samples were separated by SDS-PAGE, and immunoblotted as described. .. CEM-T4 T cells were starved for 20 min in methionine-free, cysteine-free RPMI/5% dialysed FCS (Invitrogen), labeled with [35 S]methionine/[35 S]cysteine (EasyTag EXPRESS, PerkinElmer) for 15 min, then chased in RPMI/10% FCS at 37°C. .. Cells were lysed in 1% Triton X-100 at the indicated timepoints, and subjected to immunoprecipitation with anti-PPP2R5D as described.

    Article Title: Altered intracellular calcium homeostasis and endoplasmic reticulum redox state in Saccharomyces cerevisiae cells lacking Grx6 glutaredoxin
    Article Snippet: Paragraph title: Pulse-chase labeling and immunoprecipitation of CPY ... Samples (∼6 × 108 cells) from cultures in SC medium were resuspended in 10 ml of the same medium lacking methionine and cysteine and incubated for 30 min at the appropriate temperature before adding 500 μCi of a [35 S]methionine/[35 S]cysteine cocktail (Perkin Elmer, Waltham, MA).

    Article Title: Mitochondrial translation requires folate-dependent tRNA methylation
    Article Snippet: Then media was changed to DMEM with 10 % dFBS without methionine (MP Biomedicals). .. Following a 30 min incubation, cytosolic translation was inhibited by emetine hydrochloride (0.1 mg/ml, Sigma-Aldrich) and labeling was conducted for one hour after the addition of 500 μCi [35 S]-methionine (EasyTag™ L[ S]-Methionine, Perkin Elmer). .. Protein lysates (30 μg) were then separated on a 15% polyacrylamide gel (8.3 × 7.3 cm) and dried using a 443 Slab Dryer (BioRad).

    Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
    Article Snippet: Briefly, cells were grown in 100-mm-diameter dishes and transfected with the indicated plasmids. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM). .. Harvested cells were lysed, and PRLR proteins were immunoprecipitated with Flag or PRLR antibody, separated on SDS-PAGE gel, and analyzed by autoradiography.

    Article Title: The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis
    Article Snippet: Briefly WT and Top1mt -KO MEFs were washed with methionine/cysteine-free DMEM, 2 mM l -glutamine, 96 μg/mL cysteine and 5% (v/v) dialyzed FBS and incubated for 10 min in the same media at 37 °C. .. 100 μg/mL emetine was then added directly to the media in order to inhibit cytosolic translation; after 20 min proteins newly synthetized in the mitochondrial compartment were labeled with 100 μCi [35 S]-methionine (Perkin Elmer) for 1 h at 37 °C. .. Cells were then washed three times with PBS and lysed in PBS, 0.1% n -dodecyl-β-d -maltoside (DDM, Sigma), 1% SDS, 1X protease inhibitor cocktail (Roche) and 50 U benzonase (Novagen).

    Article Title: Reduced phosphorylation of ribosomal protein S6 is associated with sensitivity to MEK inhibition in gastric cancer cells
    Article Snippet: Finally, the signals were detected using an ECL Western blotting analysis system (GE Healthcare, Piscataway, NJ, USA) in accordance with the manufacturer's instructions. .. To determine the rate of protein synthesis, cells treated with DMSO or 1 μM PD0325901 were incubated with methionine‐free RPMI‐1640 for 10 min, and then labeled with 2 MBq/mL [35 S]methionine (PerkinElmer Japan, Tokyo, Japan) for 20 min. After a wash with PBS, the cells were lysed with a Total Protein Extraction Kit (BioChain, Hayward, CA, USA) in accordance with the manufacturer's instructions and centrifuged at 17 400 g for 20 min. .. The resulting cell lysates (40 μg/sample) were resuspended in Laemmli sample buffer and subjected to SDS‐PAGE using 10% gel.

    Article Title: Downregulation of ribosome biogenesis during early forebrain development
    Article Snippet: Paragraph title: 35 S-Methionine labeling ... Cells were serum starved in methionine-free DMEM for 1 hr at 37°C, then incubated with 51 μCi 35 S-Methionine (Perkin Elmer NEG709A) at 37°C for an additional hour.

    Article Title: Non-canonical translation initiation of the spliced mRNA encoding the human T-cell leukemia virus type 1 basic leucine zipper protein
    Article Snippet: Other monocistronic mRNAs were translated in RRL standard salt conditions (40 mM KOAc and 0.25 mM MgOAc2 ). .. All translation reaction mixtures were incubated at 30°C for 90 min. For 35 S-methionine labeling, the RRL (Flexi® -RRL; #L4540; Promega) reactions were performed as described in , using 0.02 mM of amino acids lacking methionine and supplemented with 0.6 mCi/ ml [35 S]-methionine (PerkinElmer, Inc., Waltham, MA, USA). .. The translation reaction was stopped with 90 μl of protein loading buffer (10% sodium dodecyl sulphate, 50 mM Tris (pH 6.8), 10% glycerol, 100 mM DTT and 0.1% bromophenol blue) and 10 μl of the final reaction was resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (12%), and the labeled products were visualized and quantified using a Storm PhosphorImager (GE Healthcare) as specified in ( ).

    Article Title: Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries
    Article Snippet: Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]). .. Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]).

    Article Title: Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication
    Article Snippet: Pulse-labeling of HepG2 cells with [35 S]methionine was performed as previously described ( ). .. Cells were treated with drugs as described above for 24 h before they were washed with media lacking methionine for 1 h. Cultures were then pulsed with [35 S]methionine (200 µCi/well EasyTag express protein labeling mix; PerkinElmer) for 45 min, lysed, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). .. The gel was dried and exposed to phosphor screen for 24 h before being imaged on a Bio-Rad personal molecular imager system.

    Article Title: Paired Ig-Like Type 2 Receptor-Derived Agonist Ligands Ameliorate Inflammatory Reactions by Downregulating β1 Integrin Activity
    Article Snippet: Mouse WEHI274.1 monocytes were suspended and incubated in methionine-free medium to deplete intracellular methionine for 15 min at 37°C under 5% CO2 . .. Labeling medium containing 0.2 mCi/ml of [35 S]-methionine (PerkinElmer Inc., USA) was added, followed by incubation for 30 min at 37°C. .. Methionine-containing medium was added into the cell suspension.

    Purification:

    Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
    Article Snippet: For the in vitro ubiquitination assay, GST-PRLR proteins were expressed in bacteria, purified by using glutathione beads, and preincubated (1 μg) with 25-μg extracts from 293T cells treated with PRL (200 ng/ml for 20 min) in a kinase buffer containing 50 mM Tris HCl (pH 7.5), 5 mM MgCl2 , 0.5 mM dithiothreitol, 5 mM NaF, 10 nM okadaic acid, 50 μM ATP, and 10 μM [32 P-γ]ATP for 30 min at 30°C. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

    Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
    Article Snippet: His-tagged MetRS was purified from Escherichia coli XL1 Blue cells (Agilent) carrying the E. coli methionyl-tRNA synthetase (MetRS) expression plasmid pRA101 ( ) as described previously ( ). .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ). .. For measurements of tRNA binding, seven 2-fold serial dilutions of eIF2 with final concentrations from 3.125 to 200 nM were incubated for 15 min at 26°C in Binding Assay Buffer (25 mM HEPES-KOH (pH 7.6), 2.5 mM Mg(OAc)2 , 80 mM KOAc (pH 7.6), 2 mM DTT and 0.5 mM guanosine 5′-[β,γ-imido]triphosphate (GDPNP)-Mg2+ ).

    Article Title: Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis
    Article Snippet: Paragraph title: E. coli MetRS purification and aminoacylation of initiator tRNAMet ... Aminoacylation reactions containing 5 μM tRNAi Met , 2 mM ATP, 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS in 1× reaction buffer A (100 mM HEPES–KOH [pH 7.5], 10 mM KCl and 1 mM DTT) were incubated at 37°C for 30 min.

    Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
    Article Snippet: The Δ2A versions of P1 were designed to encode the first 12 bases of the 2A protein-coding region followed by a His6 tag to allow purification in other experiments. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

    Polymerase Chain Reaction:

    Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
    Article Snippet: A plasmid encoding the capsid and 3C protease genome regions of the O1 Manisa strain of FMDV in pBG200 T7 expression vectors ( ) were used as a template to generate the plasmids pBG200-P1-2A and pBG200-P1-Δ2A, with the primer sequences described in , by PCR. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

    Protein Extraction:

    Article Title: Reduced phosphorylation of ribosomal protein S6 is associated with sensitivity to MEK inhibition in gastric cancer cells
    Article Snippet: Finally, the signals were detected using an ECL Western blotting analysis system (GE Healthcare, Piscataway, NJ, USA) in accordance with the manufacturer's instructions. .. To determine the rate of protein synthesis, cells treated with DMSO or 1 μM PD0325901 were incubated with methionine‐free RPMI‐1640 for 10 min, and then labeled with 2 MBq/mL [35 S]methionine (PerkinElmer Japan, Tokyo, Japan) for 20 min. After a wash with PBS, the cells were lysed with a Total Protein Extraction Kit (BioChain, Hayward, CA, USA) in accordance with the manufacturer's instructions and centrifuged at 17 400 g for 20 min. .. The resulting cell lysates (40 μg/sample) were resuspended in Laemmli sample buffer and subjected to SDS‐PAGE using 10% gel.

    Staining:

    Article Title: Mitochondrial translation requires folate-dependent tRNA methylation
    Article Snippet: Following a 30 min incubation, cytosolic translation was inhibited by emetine hydrochloride (0.1 mg/ml, Sigma-Aldrich) and labeling was conducted for one hour after the addition of 500 μCi [35 S]-methionine (EasyTag™ L[ S]-Methionine, Perkin Elmer). .. Following a 30 min incubation, cytosolic translation was inhibited by emetine hydrochloride (0.1 mg/ml, Sigma-Aldrich) and labeling was conducted for one hour after the addition of 500 μCi [35 S]-methionine (EasyTag™ L[ S]-Methionine, Perkin Elmer).

    Article Title: In vitro centromere and kinetochore assembly on defined chromatin templates
    Article Snippet: Human and Xenopus CENP-C was in vitro translated (IVT) in rabbit reticulocyte extracts in the presence of 10 mCi/Ml [35 S]methionine (Perkin Elmer) using the TnT Quick-Coupled Transcription/Translation system (Promega) according to the manufacturer’s instructions. .. Reactions were incubated at 4°C for 1 h. The beads were washed three times with bead buffer and resuspended in 4x SDS loading buffer.

    Mouse Assay:

    Article Title: Paired Ig-Like Type 2 Receptor-Derived Agonist Ligands Ameliorate Inflammatory Reactions by Downregulating β1 Integrin Activity
    Article Snippet: Labeling medium containing 0.2 mCi/ml of [35 S]-methionine (PerkinElmer Inc., USA) was added, followed by incubation for 30 min at 37°C. .. Labeling medium containing 0.2 mCi/ml of [35 S]-methionine (PerkinElmer Inc., USA) was added, followed by incubation for 30 min at 37°C.

    SDS Page:

    Article Title: Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants
    Article Snippet: CEM-T4 T cells were starved for 20 min in methionine-free, cysteine-free RPMI/5% dialysed FCS (Invitrogen), labeled with [35 S]methionine/[35 S]cysteine (EasyTag EXPRESS, PerkinElmer) for 15 min, then chased in RPMI/10% FCS at 37°C. .. Cells were lysed in 1% Triton X-100 at the indicated timepoints, and subjected to immunoprecipitation with anti-PPP2R5D as described.

    Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
    Article Snippet: Ubiquitination reactions were carried out with a total volume of 20 μl containing 2 μg of ubiquitin, 20 pmol of E1, and 100 pmol of E2 as well as 2 mM ATP at 37°C for 60 min. Boiling in SDS-sample buffer terminated the reactions, and the products were analyzed by SDS-PAGE and autoradiography. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

    Article Title: The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis
    Article Snippet: 100 μg/mL emetine was then added directly to the media in order to inhibit cytosolic translation; after 20 min proteins newly synthetized in the mitochondrial compartment were labeled with 100 μCi [35 S]-methionine (Perkin Elmer) for 1 h at 37 °C. .. Cells were then washed three times with PBS and lysed in PBS, 0.1% n -dodecyl-β-d -maltoside (DDM, Sigma), 1% SDS, 1X protease inhibitor cocktail (Roche) and 50 U benzonase (Novagen).

    Article Title: In vitro centromere and kinetochore assembly on defined chromatin templates
    Article Snippet: Human and Xenopus CENP-C was in vitro translated (IVT) in rabbit reticulocyte extracts in the presence of 10 mCi/Ml [35 S]methionine (Perkin Elmer) using the TnT Quick-Coupled Transcription/Translation system (Promega) according to the manufacturer’s instructions. .. Reactions were incubated at 4°C for 1 h. The beads were washed three times with bead buffer and resuspended in 4x SDS loading buffer.

    Article Title: Adult-onset obesity is triggered by impaired mitochondrial gene expression
    Article Snippet: Mitochondria were supplemented with 150 μCi of [35 S]methionine (PerkinElmer) for 60 min at 37°C. .. After labeling, mitochondria were washed in translation buffer and suspended in radioimmunoprecipitation assay lysis buffer.

    Article Title: The Rab6-regulated KIF1C kinesin motor domain contributes to Golgi organization
    Article Snippet: Full-length myc-KIF1C synthesized in vitro with 35 S-methionine (EasyTag, Perkin Elmer, San Jose, CA) was desalted and incubated with GTPγS-preloaded Rabs (4.2 μM) for 1 hr in Buffer A (−BSA), 1 mM DTT, 2.5 mM ADP, 0.5 mM GTPγS. .. Microtubules (0.8 mg/ml), polymerized in 80 mM PIPES, 1 mM MgCl2 , 1 mM EGTA, pH 6.8 (BRB80), 1 mM DTT, 1 mM GTP, 10% DMSO, spun through a 40% glycerol cushion, and resuspended in BRB80, 1 mM DTT, 0.2 mM Paclitaxel (Cytoskeleton, Inc.), were incubated with the KIF1C-Rab complexes for 1 hr before being spun through a 10% sucrose, 20 µM Paclitaxel, 1 mM DTT, at 65K for 5 min (Optima TLX, Beckman Coulter, Inc., Indianapolis, IN).

    Article Title: Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA
    Article Snippet: Cells were then cultured in a medium containing 0.15 mCi ml–1 35 S-methionine (Perkin Elmer, Boston, USA) for 1 h and collected. .. Cells were then cultured in a medium containing 0.15 mCi ml–1 35 S-methionine (Perkin Elmer, Boston, USA) for 1 h and collected.

    Article Title: Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication
    Article Snippet: Pulse-labeling of HepG2 cells with [35 S]methionine was performed as previously described ( ). .. Cells were treated with drugs as described above for 24 h before they were washed with media lacking methionine for 1 h. Cultures were then pulsed with [35 S]methionine (200 µCi/well EasyTag express protein labeling mix; PerkinElmer) for 45 min, lysed, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). .. The gel was dried and exposed to phosphor screen for 24 h before being imaged on a Bio-Rad personal molecular imager system.

    Plasmid Preparation:

    Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
    Article Snippet: His-tagged MetRS was purified from Escherichia coli XL1 Blue cells (Agilent) carrying the E. coli methionyl-tRNA synthetase (MetRS) expression plasmid pRA101 ( ) as described previously ( ). .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ).

    Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
    Article Snippet: Radiolabeled P1-2A was generated from plasmids using in vitro transcription and translation reactions in rabbit reticulocyte lysates (TnT quick; Promega) according to the manufacturer's instructions. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS. .. Products of processing reactions were separated by SDS-PAGE.

    In Vitro:

    Article Title: Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase
    Article Snippet: For the in vitro ubiquitination assay, GST-PRLR proteins were expressed in bacteria, purified by using glutathione beads, and preincubated (1 μg) with 25-μg extracts from 293T cells treated with PRL (200 ng/ml for 20 min) in a kinase buffer containing 50 mM Tris HCl (pH 7.5), 5 mM MgCl2 , 0.5 mM dithiothreitol, 5 mM NaF, 10 nM okadaic acid, 50 μM ATP, and 10 μM [32 P-γ]ATP for 30 min at 30°C. .. After starvation in DMEM lacking methionine and cysteine and metabolic labeling with a [35 S]methionine-[35 S]cysteine mixture (Perkin-Elmer), cells were harvested at each chase time point with complete DMEM supplemented with FBS (0.5%), PRL (20 ng/ml), and unlabeled methionine and cysteine (2 mM).

    Article Title: MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2
    Article Snippet: Yeast tRNAi Met was synthesized by in vitro transcription with T7 polymerase as described previously ( ). .. Aminoacylation reactions with 5 μM synthesized tRNAi Met , 2 mM adenosine triphosphate-Mg2+ , 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS were incubated in 1× Reaction Buffer (100 mM HEPES-KOH (pH 7.5), 1 mM DTT and 10 mM KCl) for 30 min at 30°C, and aminoacylated Met-tRNAi Met was purified by phenol/chloroform extraction ( ).

    Article Title: In vitro centromere and kinetochore assembly on defined chromatin templates
    Article Snippet: Actin antibodies were (provided by Julie Theriot, Stanford University, Stanford, USA) and α-CyclinB was purchased from Santa Cruz Biotechnology. .. Human and Xenopus CENP-C was in vitro translated (IVT) in rabbit reticulocyte extracts in the presence of 10 mCi/Ml [35 S]methionine (Perkin Elmer) using the TnT Quick-Coupled Transcription/Translation system (Promega) according to the manufacturer’s instructions. .. For a binding reaction (60μl total volume), 5 μl of each IVT protein were mixed with chromatin arrays in bead buffer (75mM Tris-HCl pH 7.5, 50mM NaCl, 0.25mM EDTA, 0.05% Triton-X-100).

    Article Title: Non-canonical translation initiation of the spliced mRNA encoding the human T-cell leukemia virus type 1 basic leucine zipper protein
    Article Snippet: Paragraph title: In vitro translation ... All translation reaction mixtures were incubated at 30°C for 90 min. For 35 S-methionine labeling, the RRL (Flexi® -RRL; #L4540; Promega) reactions were performed as described in , using 0.02 mM of amino acids lacking methionine and supplemented with 0.6 mCi/ ml [35 S]-methionine (PerkinElmer, Inc., Waltham, MA, USA).

    Article Title: The Rab6-regulated KIF1C kinesin motor domain contributes to Golgi organization
    Article Snippet: The eluate was diluted fivefold with Buffer C (30 mM Hepes, pH 7.4, 1 mM MgCl2 , 1 mM EGTA, 25 µM ATP) and applied to HiTrap Q FF (GE Healthcare), washed with Buffer C + 100 mM NaCl, before being eluted by gradient to 500 mM NaCl in Buffer C. Binding of the purified components was assayed in the same manner as in vitro translation-synthesized constructs. .. Full-length myc-KIF1C synthesized in vitro with 35 S-methionine (EasyTag, Perkin Elmer, San Jose, CA) was desalted and incubated with GTPγS-preloaded Rabs (4.2 μM) for 1 hr in Buffer A (−BSA), 1 mM DTT, 2.5 mM ADP, 0.5 mM GTPγS. .. Microtubules (0.8 mg/ml), polymerized in 80 mM PIPES, 1 mM MgCl2 , 1 mM EGTA, pH 6.8 (BRB80), 1 mM DTT, 1 mM GTP, 10% DMSO, spun through a 40% glycerol cushion, and resuspended in BRB80, 1 mM DTT, 0.2 mM Paclitaxel (Cytoskeleton, Inc.), were incubated with the KIF1C-Rab complexes for 1 hr before being spun through a 10% sucrose, 20 µM Paclitaxel, 1 mM DTT, at 65K for 5 min (Optima TLX, Beckman Coulter, Inc., Indianapolis, IN).

    Article Title: Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis
    Article Snippet: For aminoacylation of yeast initiator tRNAi Met , E. coli MetRS was purified from E. coli strain XL1 Blue (Agilent) transformed with pRA101 ( ) and yeast tRNAi Met was prepared by in vitro transcription as described previously ( ). .. Aminoacylation reactions containing 5 μM tRNAi Met , 2 mM ATP, 0.3 μM [35 S]methionine (Perkin Elmer), 10 mM MgCl2 and 1 μM MetRS in 1× reaction buffer A (100 mM HEPES–KOH [pH 7.5], 10 mM KCl and 1 mM DTT) were incubated at 37°C for 30 min.

    Article Title: The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers
    Article Snippet: Radiolabeled P1-2A was generated from plasmids using in vitro transcription and translation reactions in rabbit reticulocyte lysates (TnT quick; Promega) according to the manufacturer's instructions. .. Reaction mixtures contained 20 ng/μl plasmid DNA, 8 Bq/μl [35 S]methionine (EasyTag; PerkinElmer), and various concentrations of hsp90 inhibitor and were incubated at 30°C for 1.5 h. Processing and assembly of P1-2A into pentamers was achieved by the addition of 1 μM 3Cpro to the reaction and incubation at 37°C for 1 h. Following incubation, free radiolabel was removed from reactions by dialysis at 4°C against phosphate-buffered saline (PBS), using mini-dialysis units (Slide-A-Lyzer; Pierce) that had been preblocked with 1% bovine serum albumin in PBS.

    Radio Immunoprecipitation:

    Article Title: Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries
    Article Snippet: Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]). .. Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]).

    Immunoprecipitation:

    Article Title: Altered intracellular calcium homeostasis and endoplasmic reticulum redox state in Saccharomyces cerevisiae cells lacking Grx6 glutaredoxin
    Article Snippet: Paragraph title: Pulse-chase labeling and immunoprecipitation of CPY ... Samples (∼6 × 108 cells) from cultures in SC medium were resuspended in 10 ml of the same medium lacking methionine and cysteine and incubated for 30 min at the appropriate temperature before adding 500 μCi of a [35 S]methionine/[35 S]cysteine cocktail (Perkin Elmer, Waltham, MA).

    Article Title: Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA
    Article Snippet: Cells were then cultured in a medium containing 0.15 mCi ml–1 35 S-methionine (Perkin Elmer, Boston, USA) for 1 h and collected. .. Cells were then cultured in a medium containing 0.15 mCi ml–1 35 S-methionine (Perkin Elmer, Boston, USA) for 1 h and collected.

    Lysis:

    Article Title: Altered intracellular calcium homeostasis and endoplasmic reticulum redox state in Saccharomyces cerevisiae cells lacking Grx6 glutaredoxin
    Article Snippet: Samples (∼6 × 108 cells) from cultures in SC medium were resuspended in 10 ml of the same medium lacking methionine and cysteine and incubated for 30 min at the appropriate temperature before adding 500 μCi of a [35 S]methionine/[35 S]cysteine cocktail (Perkin Elmer, Waltham, MA). .. Samples (∼6 × 108 cells) from cultures in SC medium were resuspended in 10 ml of the same medium lacking methionine and cysteine and incubated for 30 min at the appropriate temperature before adding 500 μCi of a [35 S]methionine/[35 S]cysteine cocktail (Perkin Elmer, Waltham, MA).

    Article Title: Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries
    Article Snippet: Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]). .. Mock-infected and HCMV-infected cells were incubated for 30 min in l -methionine-free DMEM supplemented with 10% dialyzed fetal bovine serum (dFBS) and emetine (100 µg/ml) to block cytosolic translation and 250 µCi/ml [35 S]methionine (EasyTag l -[35 S]methionine [PerkinElmer, Waltham, MA]).

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    PerkinElmer adomet
    Substrate specificity and product pattern of <t>MLL3</t> protein variants. H3K4 unmethylated (H3; 1 to 19) and monomethylated H3 (1 to 17) peptides were methylated by MLL3-SET wild-type and MLL3-SET Y4884C mutant protein in the presence of complex members using unlabeled <t>AdoMet.</t> The samples were collected at different time points, and methylation was analyzed by mass spectrometry using the relative areas of the corresponding unmethylated and methylated peaks. (A) Matrix-assisted laser desorption/ionization (MALDI) spectra of the methylation of the H3K4me0 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,423.4 Da (H3K4me0), 2,437.4 Da (H3K4me1), 2,451.4 Da (H3K4me2), and 2,465.4 Da (H3K4me3). (B) MALDI spectra of the methylation of the H3K4me1 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,181.2 Da (H3K4me1), 2,195.2 Da (H3K4me2), and 2,209.2 Da (H3K4me3).
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    Substrate specificity and product pattern of MLL3 protein variants. H3K4 unmethylated (H3; 1 to 19) and monomethylated H3 (1 to 17) peptides were methylated by MLL3-SET wild-type and MLL3-SET Y4884C mutant protein in the presence of complex members using unlabeled AdoMet. The samples were collected at different time points, and methylation was analyzed by mass spectrometry using the relative areas of the corresponding unmethylated and methylated peaks. (A) Matrix-assisted laser desorption/ionization (MALDI) spectra of the methylation of the H3K4me0 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,423.4 Da (H3K4me0), 2,437.4 Da (H3K4me1), 2,451.4 Da (H3K4me2), and 2,465.4 Da (H3K4me3). (B) MALDI spectra of the methylation of the H3K4me1 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,181.2 Da (H3K4me1), 2,195.2 Da (H3K4me2), and 2,209.2 Da (H3K4me3).

    Journal: Clinical Epigenetics

    Article Title: Somatic cancer mutations in the MLL3-SET domain alter the catalytic properties of the enzyme

    doi: 10.1186/s13148-015-0075-3

    Figure Lengend Snippet: Substrate specificity and product pattern of MLL3 protein variants. H3K4 unmethylated (H3; 1 to 19) and monomethylated H3 (1 to 17) peptides were methylated by MLL3-SET wild-type and MLL3-SET Y4884C mutant protein in the presence of complex members using unlabeled AdoMet. The samples were collected at different time points, and methylation was analyzed by mass spectrometry using the relative areas of the corresponding unmethylated and methylated peaks. (A) Matrix-assisted laser desorption/ionization (MALDI) spectra of the methylation of the H3K4me0 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,423.4 Da (H3K4me0), 2,437.4 Da (H3K4me1), 2,451.4 Da (H3K4me2), and 2,465.4 Da (H3K4me3). (B) MALDI spectra of the methylation of the H3K4me1 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,181.2 Da (H3K4me1), 2,195.2 Da (H3K4me2), and 2,209.2 Da (H3K4me3).

    Article Snippet: The arrays were then incubated for 60 min at room temperature in methylation buffer containing 50 nM MLL3-SET variants and 0.76 μM radioactively labeled AdoMet (PerkinElmer, Waltham, MA, USA) and treated as described previously [ , ].

    Techniques: Methylation, Mutagenesis, Mass Spectrometry

    Activity of MLL3 protein variants. Recombinant histone H3 protein was methylated with radioactively labeled AdoMet by MLL3-SET wild-type and MLL3-SET mutant proteins either alone or in the presence of complex member proteins. (A) Example of an autoradiographic image of the SDS polyacrylamide gel. The methylation signal of H3 is indicated. (B) Quantitative analysis of the averages of duplicate experiments. The error bars indicate the standard error of the mean.

    Journal: Clinical Epigenetics

    Article Title: Somatic cancer mutations in the MLL3-SET domain alter the catalytic properties of the enzyme

    doi: 10.1186/s13148-015-0075-3

    Figure Lengend Snippet: Activity of MLL3 protein variants. Recombinant histone H3 protein was methylated with radioactively labeled AdoMet by MLL3-SET wild-type and MLL3-SET mutant proteins either alone or in the presence of complex member proteins. (A) Example of an autoradiographic image of the SDS polyacrylamide gel. The methylation signal of H3 is indicated. (B) Quantitative analysis of the averages of duplicate experiments. The error bars indicate the standard error of the mean.

    Article Snippet: The arrays were then incubated for 60 min at room temperature in methylation buffer containing 50 nM MLL3-SET variants and 0.76 μM radioactively labeled AdoMet (PerkinElmer, Waltham, MA, USA) and treated as described previously [ , ].

    Techniques: Activity Assay, Recombinant, Methylation, Labeling, Mutagenesis

    Substrate specificity of MLL3 protein variants. H3 (1 to 15) peptide arrays containing H3K4 at different methylation states and a K4A peptide were methylated with MLL3-SET wild-type and mutant proteins in the presence of complex members using radioactively labeled AdoMet. (A) Example of an autoradiographic image of the methylated peptide SPOT arrays, peptides with the corresponding lysine variants are indicated. (B) Quantitative analysis of the average methylation signals of two independent experiments. The methylation data were normalized to the H3K4me0 methylation signal obtained with the individual MLL3-SET variants. The error bars indicate the standard error of the mean.

    Journal: Clinical Epigenetics

    Article Title: Somatic cancer mutations in the MLL3-SET domain alter the catalytic properties of the enzyme

    doi: 10.1186/s13148-015-0075-3

    Figure Lengend Snippet: Substrate specificity of MLL3 protein variants. H3 (1 to 15) peptide arrays containing H3K4 at different methylation states and a K4A peptide were methylated with MLL3-SET wild-type and mutant proteins in the presence of complex members using radioactively labeled AdoMet. (A) Example of an autoradiographic image of the methylated peptide SPOT arrays, peptides with the corresponding lysine variants are indicated. (B) Quantitative analysis of the average methylation signals of two independent experiments. The methylation data were normalized to the H3K4me0 methylation signal obtained with the individual MLL3-SET variants. The error bars indicate the standard error of the mean.

    Article Snippet: The arrays were then incubated for 60 min at room temperature in methylation buffer containing 50 nM MLL3-SET variants and 0.76 μM radioactively labeled AdoMet (PerkinElmer, Waltham, MA, USA) and treated as described previously [ , ].

    Techniques: Methylation, Mutagenesis, Labeling

    Structure of the MLL1-SET domain bound to the H3 peptide and cofactor product S-adenosyl-L-homocysteine (AdoHcy) (pdb code 2W5Y). Note that an MLL3 structure currently is not available. (A) The protein is shown as blue ribbon, and the peptide is shown in orange in the stick model with the target nitrogen atom colored white. The residues corresponding to Asn4848 and Tyr4884 are displayed in red and green, respectively, the corresponding alignment of MLL1 and MLL3 is shown in Additional file 1 : Figure S4. (B) Details of the MLL1-SET structure showing that N3906 (corresponding to N4848) is involved in an H-bond to AdoMet (shown in stick model with coloring by atom type). (C) Details of the MLL1-SET structures showing the hydrophobic and aromatic pocket of MLL1 surrounding the target nitrogen atom which consists of Y3942 (corresponding to Y4884, shown in green) and Y4800, I4847, and Y4885 (all designations refer to MLL3, residues shown in blue).

    Journal: Clinical Epigenetics

    Article Title: Somatic cancer mutations in the MLL3-SET domain alter the catalytic properties of the enzyme

    doi: 10.1186/s13148-015-0075-3

    Figure Lengend Snippet: Structure of the MLL1-SET domain bound to the H3 peptide and cofactor product S-adenosyl-L-homocysteine (AdoHcy) (pdb code 2W5Y). Note that an MLL3 structure currently is not available. (A) The protein is shown as blue ribbon, and the peptide is shown in orange in the stick model with the target nitrogen atom colored white. The residues corresponding to Asn4848 and Tyr4884 are displayed in red and green, respectively, the corresponding alignment of MLL1 and MLL3 is shown in Additional file 1 : Figure S4. (B) Details of the MLL1-SET structure showing that N3906 (corresponding to N4848) is involved in an H-bond to AdoMet (shown in stick model with coloring by atom type). (C) Details of the MLL1-SET structures showing the hydrophobic and aromatic pocket of MLL1 surrounding the target nitrogen atom which consists of Y3942 (corresponding to Y4884, shown in green) and Y4800, I4847, and Y4885 (all designations refer to MLL3, residues shown in blue).

    Article Snippet: The arrays were then incubated for 60 min at room temperature in methylation buffer containing 50 nM MLL3-SET variants and 0.76 μM radioactively labeled AdoMet (PerkinElmer, Waltham, MA, USA) and treated as described previously [ , ].

    Techniques:

    Product specificity of MLL3-SET wild-type and Y4884C at protein level. (A) Methylation of recombinant histone H3 protein by MLL3-SET wild-type and Y4884C alone and in the presence of complex members using unlabeled AdoMet as cofactor. After methylation, the proteins were separated by SDS-PAGE, blotted and the methylation was detected using an H3K4 trimethylated antibody (upper panel). The lower panel shows the Ponceau S-stained image of the blot. The bar diagram shows the average methylation signal from two independent experiments. The error bars indicate the standard error of the mean. (B) Global histone H3K4me3 methylation analysis from HEK293 cells. The cells were transfected with different MLL3 variant plasmids, histones were isolated, and H3K4me3 methylation was probed by Western blot. The upper image shows the H3K4me3 signal and a Ponceau S stain as loading control. The bar diagram shows the quantification from three experiments. The error bars display the standard error of the mean. The signal obtained from the MLL3 was set to 1, and the other signals were normalized accordingly.

    Journal: Clinical Epigenetics

    Article Title: Somatic cancer mutations in the MLL3-SET domain alter the catalytic properties of the enzyme

    doi: 10.1186/s13148-015-0075-3

    Figure Lengend Snippet: Product specificity of MLL3-SET wild-type and Y4884C at protein level. (A) Methylation of recombinant histone H3 protein by MLL3-SET wild-type and Y4884C alone and in the presence of complex members using unlabeled AdoMet as cofactor. After methylation, the proteins were separated by SDS-PAGE, blotted and the methylation was detected using an H3K4 trimethylated antibody (upper panel). The lower panel shows the Ponceau S-stained image of the blot. The bar diagram shows the average methylation signal from two independent experiments. The error bars indicate the standard error of the mean. (B) Global histone H3K4me3 methylation analysis from HEK293 cells. The cells were transfected with different MLL3 variant plasmids, histones were isolated, and H3K4me3 methylation was probed by Western blot. The upper image shows the H3K4me3 signal and a Ponceau S stain as loading control. The bar diagram shows the quantification from three experiments. The error bars display the standard error of the mean. The signal obtained from the MLL3 was set to 1, and the other signals were normalized accordingly.

    Article Snippet: The arrays were then incubated for 60 min at room temperature in methylation buffer containing 50 nM MLL3-SET variants and 0.76 μM radioactively labeled AdoMet (PerkinElmer, Waltham, MA, USA) and treated as described previously [ , ].

    Techniques: Methylation, Recombinant, SDS Page, Staining, Transfection, Variant Assay, Isolation, Western Blot

    METTL21B-mediated methylation of eEF1A in vitro is GTP- and aminoacyl-tRNA-dependent. ( A ) Scheme for partial purification of eEF1A from extracts by cation exchange (S-column)-based chromatography. 0.3S fraction; material eluted from S-column with 0.3 M NaCl. ( B ) Nucleotide dependency of METTL21B-mediated methylation of eEF1A from HeLa cells. Incorporation of [ 3 H]-methyl into eEF1A from the HeLa 0.3S fraction incubated with [ 3 H]AdoMet, METTL21B and indicated nucleotides, was assessed by fluorography (upper panel) of Ponceau S-stained membrane (lower panel). Arrows indicate the position of eEF1A and METTL21B. ( C ) Nucleotide dependency of METTL21B-mediated methylation of recombinant eEF1A1. Incorporation of [ 3 H]-methyl into recombinant eEF1A1 incubated with [ 3 H]AdoMet, METTL21B and indicated nucleotides, was assessed by fluorography (upper panel). ( D ) METTL21B-mediated methylation of eEF1A from HeLa cells is RNA-dependent. Incorporation of [ 3 H]-methyl into eEF1A from the HeLa 0.3S fraction incubated with [ 3 H]AdoMet, GTP and METTL21B, in the absence or presence of RNAse A, was assessed by fluorography (upper panel). ( E ) METTL21B-mediated methylation of HeLa-derived eEF1A is GTP-dependent and stimulated by the addition of aminoacyl-tRNA. Incorporation of [ 3 H]-methyl into eEF1A from the HeLa 0.3S fraction incubated with [ 3 H]AdoMet and METTL21B, with or without GTP and in the absence or presence of (HeLa-derived) aminoacyl-tRNA or large RNAs, was assessed by fluorography (upper panel). ( F ) METTL21B-mediated methylation of HeLa-derived eEF1A is stimulated by addition of aminoacyl-tRNA. eEF1A was partially purified from extracts by consecutive Q-column/S-column-based ion-exchange chromatography, as indicated in Figure 2A . Incorporation of [ 3 H]-methyl into eEF1A from HeLa FTQ/0.3S fraction incubated with [ 3 H]AdoMet, METTL21B, GTP and increasing concentration of (HeLa-derived) aminoacyl-tRNA or deacylated tRNA, was assessed by fluorography (upper panel).

    Journal: Nucleic Acids Research

    Article Title: The novel lysine specific methyltransferase METTL21B affects mRNA translation through inducible and dynamic methylation of Lys-165 in human eukaryotic elongation factor 1 alpha (eEF1A)

    doi: 10.1093/nar/gkx002

    Figure Lengend Snippet: METTL21B-mediated methylation of eEF1A in vitro is GTP- and aminoacyl-tRNA-dependent. ( A ) Scheme for partial purification of eEF1A from extracts by cation exchange (S-column)-based chromatography. 0.3S fraction; material eluted from S-column with 0.3 M NaCl. ( B ) Nucleotide dependency of METTL21B-mediated methylation of eEF1A from HeLa cells. Incorporation of [ 3 H]-methyl into eEF1A from the HeLa 0.3S fraction incubated with [ 3 H]AdoMet, METTL21B and indicated nucleotides, was assessed by fluorography (upper panel) of Ponceau S-stained membrane (lower panel). Arrows indicate the position of eEF1A and METTL21B. ( C ) Nucleotide dependency of METTL21B-mediated methylation of recombinant eEF1A1. Incorporation of [ 3 H]-methyl into recombinant eEF1A1 incubated with [ 3 H]AdoMet, METTL21B and indicated nucleotides, was assessed by fluorography (upper panel). ( D ) METTL21B-mediated methylation of eEF1A from HeLa cells is RNA-dependent. Incorporation of [ 3 H]-methyl into eEF1A from the HeLa 0.3S fraction incubated with [ 3 H]AdoMet, GTP and METTL21B, in the absence or presence of RNAse A, was assessed by fluorography (upper panel). ( E ) METTL21B-mediated methylation of HeLa-derived eEF1A is GTP-dependent and stimulated by the addition of aminoacyl-tRNA. Incorporation of [ 3 H]-methyl into eEF1A from the HeLa 0.3S fraction incubated with [ 3 H]AdoMet and METTL21B, with or without GTP and in the absence or presence of (HeLa-derived) aminoacyl-tRNA or large RNAs, was assessed by fluorography (upper panel). ( F ) METTL21B-mediated methylation of HeLa-derived eEF1A is stimulated by addition of aminoacyl-tRNA. eEF1A was partially purified from extracts by consecutive Q-column/S-column-based ion-exchange chromatography, as indicated in Figure 2A . Incorporation of [ 3 H]-methyl into eEF1A from HeLa FTQ/0.3S fraction incubated with [ 3 H]AdoMet, METTL21B, GTP and increasing concentration of (HeLa-derived) aminoacyl-tRNA or deacylated tRNA, was assessed by fluorography (upper panel).

    Article Snippet: In this case, reaction mixtures (10 μl) contained: 1× MTase Assay Buffer, 0.5 μCi of [3 H]AdoMet ([AdoMet]total = 32.6 μM), varying concentrations of METTL21B (0–2 μM), and TAP-eEF1A (∼10 μg).

    Techniques: Methylation, In Vitro, Purification, Column Chromatography, Incubation, Staining, Recombinant, Derivative Assay, Ion Exchange Chromatography, Concentration Assay

    METTL21B methylates eEF1A1 and eEF1A2 at Lys-165. ( A ) Mutation of Lys-165 abolishes METTL21B-mediated methylation of recombinant eEF1A. Recombinant eEF1A1 or eEF1A2, either wild-type (WT) or the corresponding K165A mutants, were incubated with [ 3 H]AdoMet and GTP, without or with METTL21B, and [ 3 H]-methyl incorporation in recombinant eEF1A was assessed by fluorography (upper panel) of Ponceau S-stained membrane (lower panel). Arrows indicate the position of eEF1A and METTL21B (the auto-methylation of METTL21B gives a strong signal after long exposure). ( B ) TAP-tagged eEF1A purifies from human cells as part of a multiprotein complex. Purified TAP-tagged eEF1A1 or eEF1A2, either WT or the corresponding K165A mutants, were resolved by SDS-PAGE and stained with Coomassie Blue. Indicated bands were identified by MS, to contain predominantly: eEF1A, eEF1B, eEF1D, valyl-tRNA synthetase (ValRS), or a mixture of eEF1G and eEF1A (eEF1 A/G). ( C ) TAP-eEF1A is methylated at Lys-165 in human cells in vivo . Purified TAP-tagged eEF1A1 or eEF1A2, either WT or the corresponding K165A mutants, were analyzed by MS. Shown are the relative intensities of MS signals gated for different methylation states of eEF1A1-derived, GluC-generated peptide, encompassing residues 157–169 (left), or eEF1A2-derived, AspN-generated peptide, encompassing residues 156–167 (right), with error bars indicating the range of values from three independent experiments. Red color indicates the location of the methylated residue Lys-165 within the peptide sequences. ( D ) Mutation of Lys-165 abolishes METTL21B-mediated methylation of TAP-eEF1A in vitro . Purified TAP-tagged eEF1A1 and eEF1A2, either WT or K165A-mutated, was incubated with [ 3 H]AdoMet and GTP, with or without METTL21B and [ 3 H]-methyl incorporation in TAP-eEF1A was assessed by fluorography (upper panel). The membrane (Ponceau S-stained, lower panel) was probed with anti-SBP-tag antibody (middle panel). ( E ) Comparison of eEF1A1 and eEF1A2 with respect to METTL21B-mediated methylation in vitro . Equal amounts of purified TAP-tagged eEF1A1 and eEF1A2 (∼10 μg), either WT or the corresponding K165A mutants, were incubated with [ 3 H]AdoMet ([AdoMet] total = 32.6 μM), GTP and increasing concentrations of METTL21B, and [ 3 H]-methyl incorporation into TAP-eEF1A was assayed by scintillation counting of trichloroacetic acid-precipitated material.

    Journal: Nucleic Acids Research

    Article Title: The novel lysine specific methyltransferase METTL21B affects mRNA translation through inducible and dynamic methylation of Lys-165 in human eukaryotic elongation factor 1 alpha (eEF1A)

    doi: 10.1093/nar/gkx002

    Figure Lengend Snippet: METTL21B methylates eEF1A1 and eEF1A2 at Lys-165. ( A ) Mutation of Lys-165 abolishes METTL21B-mediated methylation of recombinant eEF1A. Recombinant eEF1A1 or eEF1A2, either wild-type (WT) or the corresponding K165A mutants, were incubated with [ 3 H]AdoMet and GTP, without or with METTL21B, and [ 3 H]-methyl incorporation in recombinant eEF1A was assessed by fluorography (upper panel) of Ponceau S-stained membrane (lower panel). Arrows indicate the position of eEF1A and METTL21B (the auto-methylation of METTL21B gives a strong signal after long exposure). ( B ) TAP-tagged eEF1A purifies from human cells as part of a multiprotein complex. Purified TAP-tagged eEF1A1 or eEF1A2, either WT or the corresponding K165A mutants, were resolved by SDS-PAGE and stained with Coomassie Blue. Indicated bands were identified by MS, to contain predominantly: eEF1A, eEF1B, eEF1D, valyl-tRNA synthetase (ValRS), or a mixture of eEF1G and eEF1A (eEF1 A/G). ( C ) TAP-eEF1A is methylated at Lys-165 in human cells in vivo . Purified TAP-tagged eEF1A1 or eEF1A2, either WT or the corresponding K165A mutants, were analyzed by MS. Shown are the relative intensities of MS signals gated for different methylation states of eEF1A1-derived, GluC-generated peptide, encompassing residues 157–169 (left), or eEF1A2-derived, AspN-generated peptide, encompassing residues 156–167 (right), with error bars indicating the range of values from three independent experiments. Red color indicates the location of the methylated residue Lys-165 within the peptide sequences. ( D ) Mutation of Lys-165 abolishes METTL21B-mediated methylation of TAP-eEF1A in vitro . Purified TAP-tagged eEF1A1 and eEF1A2, either WT or K165A-mutated, was incubated with [ 3 H]AdoMet and GTP, with or without METTL21B and [ 3 H]-methyl incorporation in TAP-eEF1A was assessed by fluorography (upper panel). The membrane (Ponceau S-stained, lower panel) was probed with anti-SBP-tag antibody (middle panel). ( E ) Comparison of eEF1A1 and eEF1A2 with respect to METTL21B-mediated methylation in vitro . Equal amounts of purified TAP-tagged eEF1A1 and eEF1A2 (∼10 μg), either WT or the corresponding K165A mutants, were incubated with [ 3 H]AdoMet ([AdoMet] total = 32.6 μM), GTP and increasing concentrations of METTL21B, and [ 3 H]-methyl incorporation into TAP-eEF1A was assayed by scintillation counting of trichloroacetic acid-precipitated material.

    Article Snippet: In this case, reaction mixtures (10 μl) contained: 1× MTase Assay Buffer, 0.5 μCi of [3 H]AdoMet ([AdoMet]total = 32.6 μM), varying concentrations of METTL21B (0–2 μM), and TAP-eEF1A (∼10 μg).

    Techniques: Mutagenesis, Methylation, Recombinant, Incubation, Staining, Purification, SDS Page, Mass Spectrometry, In Vivo, Derivative Assay, Generated, In Vitro

    Identification of eEF1A1 and eEF1A2 as likely substrates of recombinant human METTL21B. ( A ) Partial purification of METTL21B substrate from HeLa cell extracts by ion exchange chromatography. The scheme for ion exchange-based fractionation is shown on the left. HeLa extracts were incubated with [ 3 H]AdoMet, ATP and METTL21B and then fractionated. Incorporation of [ 3 H]-methyl in proteins was assessed by fluorography (right, upper panel). The band corresponding to the ∼50 kDa protein detected by fluorography was visualized by Ponceau S-staining (right, lower panel, arrow) and identified by MS to contain predominantly eEF1A1 and eEF1A2 (Table 1 ). FT, flow-through. ( B and C ) eEF1A from HeLa is methylated on Lys-165 by METTL21B in vitro . HeLa extracts, either untreated or incubated with non-radioactive AdoMet, ATP and METTL21B, were fractionated as in (A), and the ∼50 kDa substrate present in 0.3S fraction was GluC-digested (B) or AspN-digested (C) and analyzed by MS. Shown are normalized extracted-ion chromatograms gated for different methylation states of an eEF1A1-derived, GluC-generated peptide, encompassing residues 157–169 (B) or an eEF1A2-derived, AspN-generated peptide, encompassing residues 156–167 (C), from untreated or METTL21B-treated HeLa extracts. Red color indicates the location of the methylated residue Lys-165 within the peptide sequences. Percentages indicate the area under each peak relative to the total area of all peaks. A.U., arbitrary units.

    Journal: Nucleic Acids Research

    Article Title: The novel lysine specific methyltransferase METTL21B affects mRNA translation through inducible and dynamic methylation of Lys-165 in human eukaryotic elongation factor 1 alpha (eEF1A)

    doi: 10.1093/nar/gkx002

    Figure Lengend Snippet: Identification of eEF1A1 and eEF1A2 as likely substrates of recombinant human METTL21B. ( A ) Partial purification of METTL21B substrate from HeLa cell extracts by ion exchange chromatography. The scheme for ion exchange-based fractionation is shown on the left. HeLa extracts were incubated with [ 3 H]AdoMet, ATP and METTL21B and then fractionated. Incorporation of [ 3 H]-methyl in proteins was assessed by fluorography (right, upper panel). The band corresponding to the ∼50 kDa protein detected by fluorography was visualized by Ponceau S-staining (right, lower panel, arrow) and identified by MS to contain predominantly eEF1A1 and eEF1A2 (Table 1 ). FT, flow-through. ( B and C ) eEF1A from HeLa is methylated on Lys-165 by METTL21B in vitro . HeLa extracts, either untreated or incubated with non-radioactive AdoMet, ATP and METTL21B, were fractionated as in (A), and the ∼50 kDa substrate present in 0.3S fraction was GluC-digested (B) or AspN-digested (C) and analyzed by MS. Shown are normalized extracted-ion chromatograms gated for different methylation states of an eEF1A1-derived, GluC-generated peptide, encompassing residues 157–169 (B) or an eEF1A2-derived, AspN-generated peptide, encompassing residues 156–167 (C), from untreated or METTL21B-treated HeLa extracts. Red color indicates the location of the methylated residue Lys-165 within the peptide sequences. Percentages indicate the area under each peak relative to the total area of all peaks. A.U., arbitrary units.

    Article Snippet: In this case, reaction mixtures (10 μl) contained: 1× MTase Assay Buffer, 0.5 μCi of [3 H]AdoMet ([AdoMet]total = 32.6 μM), varying concentrations of METTL21B (0–2 μM), and TAP-eEF1A (∼10 μg).

    Techniques: Recombinant, Purification, Ion Exchange Chromatography, Fractionation, Incubation, Staining, Mass Spectrometry, Flow Cytometry, Methylation, In Vitro, Derivative Assay, Generated

    METTL21B is a protein MTase limited to vertebrates. ( A ) Alignment of putative METTL21B orthologs from Homo sapiens (Hs; NP_056248.2), Myotis brandtii (Ms; XP_005879111.1), Python bivittatus (Pb; XP_007432709.1), Xenopus tropicalis (Xt; NP_001016660.1) and Astyanax mexicanus (Am; XP_007248457.1). Hallmark motifs found in MTF16 members are boxed. Indicated is the position of α-helixes (green rectangles) and β-strands (orange arrows), inferred from the structure of human METTL21B (PDB 4QPN). ( B ) METTL21B-dependent protein methylation in HeLa cell extracts. Incorporation of [ 3 H]-methyl in proteins from extracts incubated with [ 3 H]AdoMet and recombinant human METTL21B, in the absence or presence of ATP, was assessed by fluorography (left panel) of Ponceau S-stained membrane (right panel). Arrows indicate the positions of the ∼50 kDa substrate and METTL21B.

    Journal: Nucleic Acids Research

    Article Title: The novel lysine specific methyltransferase METTL21B affects mRNA translation through inducible and dynamic methylation of Lys-165 in human eukaryotic elongation factor 1 alpha (eEF1A)

    doi: 10.1093/nar/gkx002

    Figure Lengend Snippet: METTL21B is a protein MTase limited to vertebrates. ( A ) Alignment of putative METTL21B orthologs from Homo sapiens (Hs; NP_056248.2), Myotis brandtii (Ms; XP_005879111.1), Python bivittatus (Pb; XP_007432709.1), Xenopus tropicalis (Xt; NP_001016660.1) and Astyanax mexicanus (Am; XP_007248457.1). Hallmark motifs found in MTF16 members are boxed. Indicated is the position of α-helixes (green rectangles) and β-strands (orange arrows), inferred from the structure of human METTL21B (PDB 4QPN). ( B ) METTL21B-dependent protein methylation in HeLa cell extracts. Incorporation of [ 3 H]-methyl in proteins from extracts incubated with [ 3 H]AdoMet and recombinant human METTL21B, in the absence or presence of ATP, was assessed by fluorography (left panel) of Ponceau S-stained membrane (right panel). Arrows indicate the positions of the ∼50 kDa substrate and METTL21B.

    Article Snippet: In this case, reaction mixtures (10 μl) contained: 1× MTase Assay Buffer, 0.5 μCi of [3 H]AdoMet ([AdoMet]total = 32.6 μM), varying concentrations of METTL21B (0–2 μM), and TAP-eEF1A (∼10 μg).

    Techniques: Mass Spectrometry, Methylation, Incubation, Recombinant, Staining

    TrmO is an AdoMet-dependent methyltransferase responsible for m 6 t 6 A formation. (A) Physical interaction of TrmO and tRNAs by gel-retardation assay. The polyacrylamide gel was stained with SYBR gold (upper panel) and Coomassie brilliant blue (lower panel). Lanes 1–4 represent tRNA Thr3 (GGU) (t 6 A37) with 0, 15, 30, and 45 pmol TrmO; lanes 5 and 6 represent tRNA Thr3 (GGU) (A37) without or with TrmO (45 pmol). Lanes 7 and 8 represent tRNA Thr4 (UGU) (t 6 A37) without or with TrmO (45 pmol). Lane 9 represents TrmO (45 pmol) only. All conditions contained 15 pmol tRNA. (B) In vitro methylation by TrmO. E. coli tRNA Thr3 (GGU) transcript bearing t 6 A37 was incubated in the presence of recombinant TrmO with (left panels) or without (right panels) AdoMet. Upper and lower panels: mass chromatograms showing doubly-charged negative ions of m 6 t 6 A-containing tetramer (Um 6 t 6 AAGp; MW 1486.24, m / z 742.11) and t 6 A-containing tetramer (Ut 6 AAGp; MW 1472.23, m / z 735.10), respectively. (C) The same experiment as in B, using E. coli tRNA Thr3 (GGU) transcript bearing A37 (without t 6 A). No methylation took place in this tRNA, even in the presence of both TrmO and AdoMet. (D) The same experiment as in B, using E . coli tRNA Thr3 (GGU) isolated from the Δ trmO strain. The peak marked with an asterisk represents the m 6 t 6 A-containing fragment derived from carryover of wild-type E . coli tRNA Thr3 (GGU) bound to the oligo DNA probe. (E) Schematic domain structure of E. coli TrmO. Scale denotes amino acid numbering. (F) Close-up view of the AdoMet-binding site in the crystal structure of A. fulgidus AF0241. Bound AdoMet is shown as a red stick. Four amino acid residues that interact with AdoMet are colored. Note that only K122 (yellow) is a residue from the other subunit. The amino acid numbers of E. coli TrmO are shown in parentheses. Possible hydrogen bonds are shown as dash lines. (G) Mutation study of trmO . The Δ trmO strain was transformed with plasmid-encoded trmO wild type or mutants. The wild-type strain (BW25113) was also transformed with the empty vector (pHSG415r). The height of the mass chromatogram for the proton adduct of m 6 t 6 A ( m/z 427) in each transformant was divided by that of m 2 A ( m/z 282), and the relative ratio was normalized to the result of Δ trmO complemented with wild-type trmO . The bar graph show the average value of three experiments, and error bars indicate the SD values. N.D., not detected.

    Journal: Nucleic Acids Research

    Article Title: Discovery of the β-barrel–type RNA methyltransferase responsible for N6-methylation of N6-threonylcarbamoyladenosine in tRNAs

    doi: 10.1093/nar/gku618

    Figure Lengend Snippet: TrmO is an AdoMet-dependent methyltransferase responsible for m 6 t 6 A formation. (A) Physical interaction of TrmO and tRNAs by gel-retardation assay. The polyacrylamide gel was stained with SYBR gold (upper panel) and Coomassie brilliant blue (lower panel). Lanes 1–4 represent tRNA Thr3 (GGU) (t 6 A37) with 0, 15, 30, and 45 pmol TrmO; lanes 5 and 6 represent tRNA Thr3 (GGU) (A37) without or with TrmO (45 pmol). Lanes 7 and 8 represent tRNA Thr4 (UGU) (t 6 A37) without or with TrmO (45 pmol). Lane 9 represents TrmO (45 pmol) only. All conditions contained 15 pmol tRNA. (B) In vitro methylation by TrmO. E. coli tRNA Thr3 (GGU) transcript bearing t 6 A37 was incubated in the presence of recombinant TrmO with (left panels) or without (right panels) AdoMet. Upper and lower panels: mass chromatograms showing doubly-charged negative ions of m 6 t 6 A-containing tetramer (Um 6 t 6 AAGp; MW 1486.24, m / z 742.11) and t 6 A-containing tetramer (Ut 6 AAGp; MW 1472.23, m / z 735.10), respectively. (C) The same experiment as in B, using E. coli tRNA Thr3 (GGU) transcript bearing A37 (without t 6 A). No methylation took place in this tRNA, even in the presence of both TrmO and AdoMet. (D) The same experiment as in B, using E . coli tRNA Thr3 (GGU) isolated from the Δ trmO strain. The peak marked with an asterisk represents the m 6 t 6 A-containing fragment derived from carryover of wild-type E . coli tRNA Thr3 (GGU) bound to the oligo DNA probe. (E) Schematic domain structure of E. coli TrmO. Scale denotes amino acid numbering. (F) Close-up view of the AdoMet-binding site in the crystal structure of A. fulgidus AF0241. Bound AdoMet is shown as a red stick. Four amino acid residues that interact with AdoMet are colored. Note that only K122 (yellow) is a residue from the other subunit. The amino acid numbers of E. coli TrmO are shown in parentheses. Possible hydrogen bonds are shown as dash lines. (G) Mutation study of trmO . The Δ trmO strain was transformed with plasmid-encoded trmO wild type or mutants. The wild-type strain (BW25113) was also transformed with the empty vector (pHSG415r). The height of the mass chromatogram for the proton adduct of m 6 t 6 A ( m/z 427) in each transformant was divided by that of m 2 A ( m/z 282), and the relative ratio was normalized to the result of Δ trmO complemented with wild-type trmO . The bar graph show the average value of three experiments, and error bars indicate the SD values. N.D., not detected.

    Article Snippet: For a tRNA mutation study (Figure ), 1 μM tRNAs bearing t6 A37 were incubated at 37°C for 5 min in a 10 μl reaction mixture containing 50 mM HEPES-KOH (pH 6.7), 100 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.1 μM TrmO, and 25 μM [14 C-methyl]AdoMet (1.74 Gbq/mmol, Perkin Elmer).

    Techniques: Electrophoretic Mobility Shift Assay, Staining, In Vitro, Methylation, Incubation, Recombinant, Isolation, Derivative Assay, Binding Assay, Mutagenesis, Transformation Assay, Plasmid Preparation

    Biosynthesis of t 6 A derivatives. In E. coli , A37 of all 13 tRNAs responsible for ANN codons is modified to t 6 A by four enzymes (YgjD, YjeE, YeaZ and YrdC), which use L -threonine, bicarbonate, and ATP as substrates. For 11 tRNAs (i.e., excluding tRNA Thr1,3 ), t 6 A37 is further dehydrated to ct 6 A by TcdA in the presence of ATP. This cyclization reaction is activated by cysteine desulfurase CsdA and sulfur acceptor protein CsdE. For tRNA Thr1,3 , t 6 A37 is methylated to form m 6 t 6 A37; this reaction is catalyzed by TrmO using AdoMet as a methyl donor.

    Journal: Nucleic Acids Research

    Article Title: Discovery of the β-barrel–type RNA methyltransferase responsible for N6-methylation of N6-threonylcarbamoyladenosine in tRNAs

    doi: 10.1093/nar/gku618

    Figure Lengend Snippet: Biosynthesis of t 6 A derivatives. In E. coli , A37 of all 13 tRNAs responsible for ANN codons is modified to t 6 A by four enzymes (YgjD, YjeE, YeaZ and YrdC), which use L -threonine, bicarbonate, and ATP as substrates. For 11 tRNAs (i.e., excluding tRNA Thr1,3 ), t 6 A37 is further dehydrated to ct 6 A by TcdA in the presence of ATP. This cyclization reaction is activated by cysteine desulfurase CsdA and sulfur acceptor protein CsdE. For tRNA Thr1,3 , t 6 A37 is methylated to form m 6 t 6 A37; this reaction is catalyzed by TrmO using AdoMet as a methyl donor.

    Article Snippet: For a tRNA mutation study (Figure ), 1 μM tRNAs bearing t6 A37 were incubated at 37°C for 5 min in a 10 μl reaction mixture containing 50 mM HEPES-KOH (pH 6.7), 100 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.1 μM TrmO, and 25 μM [14 C-methyl]AdoMet (1.74 Gbq/mmol, Perkin Elmer).

    Techniques: Modification, Methylation

    Human homolog TRMO catalyzes m 6 t 6 A formation in tRNA Ser (GCU). (A) LC/MS nucleoside analysis of wild-type E. coli (left panels) and HeLa cells (right panels). Upper panels show UV traces at 254 nm, and lower panels show mass chromatograms for the proton adduct of m 6 t 6 A ( m/z 427). (B) LC/MS analysis of RNase T 1 digested human cytoplasmic tRNA Ser (GCU). All panels are mass chromatograms for triply-charged negative ions. From upper to bottom panels, m 6 t 6 A37- and Um43-containing fragment (CUm 6 t 6 AAΨCCAUUmGp; MW 3662.51, m/z 1219.83), m 6 t 6 A-containing fragment (CUm 6 t 6 AAΨCCAUUGp; MW 3648.49, m/z 1215.16), t 6 A-containing fragment (CUt 6 AAΨCCAUUGp; MW 3634.48, m/z 1210.49) and unmodified fragment (CUAAΨCCAUUGp; MW 3489.44, m/z 1162.14). Ψ39 and Um44 are predicted from the sequence of rat tRNA Ser (GCU). (C) CID spectrum of m 6 t 6 A- and Um-containing fragment of human cytoplasmic tRNA Ser (GCU). (D) Transcript of human cytoplasmic tRNA Ser (GCU) bearing t 6 A37 used for in vitro reconstitution of m 6 t 6 A37. Arrowheads indicate cleavage sites by RNase A. (E) In vitro methylation by recombinant TRMO. Human cytoplasmic tRNA Ser (GCU) transcript bearing t 6 A37 was incubated with TRMO and AdoMet (left most panels), TRMO only (left middle panels), AdoMet only (right middle panels) or E. coli TrmO and AdoMet (right most panels). Upper and lower panels show mass chromatograms for singly charged negative ions of t 6 A-containing trimer (t 6 AAUp; MW 1127.18, m / z 1126.17) and m 6 t 6 A-containing trimer (m 6 t 6 AAUp; MW 1141.19, m / z 1140.19), respectively. (F) LC/MS nucleoside analysis of human cytoplasmic tRNA Ser (GCU) transcript bearing t 6 A37, which was incubated with TRMO and AdoMet (left most panels), TRMO only (left middle panels), AdoMet only (right middle panels), or E. coli TrmO and AdoMet (right most panels). Upper panels show UV trace at 254 nm. Middle and lower panels show mass chromatograms for the proton adduct of t 6 A ( m/z 413) and m 6 t 6 A ( m/z 427), respectively.

    Journal: Nucleic Acids Research

    Article Title: Discovery of the β-barrel–type RNA methyltransferase responsible for N6-methylation of N6-threonylcarbamoyladenosine in tRNAs

    doi: 10.1093/nar/gku618

    Figure Lengend Snippet: Human homolog TRMO catalyzes m 6 t 6 A formation in tRNA Ser (GCU). (A) LC/MS nucleoside analysis of wild-type E. coli (left panels) and HeLa cells (right panels). Upper panels show UV traces at 254 nm, and lower panels show mass chromatograms for the proton adduct of m 6 t 6 A ( m/z 427). (B) LC/MS analysis of RNase T 1 digested human cytoplasmic tRNA Ser (GCU). All panels are mass chromatograms for triply-charged negative ions. From upper to bottom panels, m 6 t 6 A37- and Um43-containing fragment (CUm 6 t 6 AAΨCCAUUmGp; MW 3662.51, m/z 1219.83), m 6 t 6 A-containing fragment (CUm 6 t 6 AAΨCCAUUGp; MW 3648.49, m/z 1215.16), t 6 A-containing fragment (CUt 6 AAΨCCAUUGp; MW 3634.48, m/z 1210.49) and unmodified fragment (CUAAΨCCAUUGp; MW 3489.44, m/z 1162.14). Ψ39 and Um44 are predicted from the sequence of rat tRNA Ser (GCU). (C) CID spectrum of m 6 t 6 A- and Um-containing fragment of human cytoplasmic tRNA Ser (GCU). (D) Transcript of human cytoplasmic tRNA Ser (GCU) bearing t 6 A37 used for in vitro reconstitution of m 6 t 6 A37. Arrowheads indicate cleavage sites by RNase A. (E) In vitro methylation by recombinant TRMO. Human cytoplasmic tRNA Ser (GCU) transcript bearing t 6 A37 was incubated with TRMO and AdoMet (left most panels), TRMO only (left middle panels), AdoMet only (right middle panels) or E. coli TrmO and AdoMet (right most panels). Upper and lower panels show mass chromatograms for singly charged negative ions of t 6 A-containing trimer (t 6 AAUp; MW 1127.18, m / z 1126.17) and m 6 t 6 A-containing trimer (m 6 t 6 AAUp; MW 1141.19, m / z 1140.19), respectively. (F) LC/MS nucleoside analysis of human cytoplasmic tRNA Ser (GCU) transcript bearing t 6 A37, which was incubated with TRMO and AdoMet (left most panels), TRMO only (left middle panels), AdoMet only (right middle panels), or E. coli TrmO and AdoMet (right most panels). Upper panels show UV trace at 254 nm. Middle and lower panels show mass chromatograms for the proton adduct of t 6 A ( m/z 413) and m 6 t 6 A ( m/z 427), respectively.

    Article Snippet: For a tRNA mutation study (Figure ), 1 μM tRNAs bearing t6 A37 were incubated at 37°C for 5 min in a 10 μl reaction mixture containing 50 mM HEPES-KOH (pH 6.7), 100 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.1 μM TrmO, and 25 μM [14 C-methyl]AdoMet (1.74 Gbq/mmol, Perkin Elmer).

    Techniques: Liquid Chromatography, Mass Spectrometry, Sequencing, In Vitro, Methylation, Recombinant, Incubation

    WDR5, RbBP5, and Ash2L are required for the H3K4 methylation activity of WRAD. A , comparison of enzymatic activity of individual WRAD components and all possible binary, ternary, and quaternary complexes. W , WDR5; R , RbBP5; A , Ash2L; D , DPY-30. Histone methyltransferase assays were conducted for a period of 8 h using [3 H]AdoMet and chicken core histones as the substrate. Quenched reactions were separated by 18% Tris-glycine SDS-PAGE and visualized with Coomassie Brilliant Blue ( upper panels ) and fluorography ( lower panels ). A no-enzyme control is shown in lane 16. B , WRAD and WRA histone methyltransferase activity as a function of enzyme concentration. Methyltransferase activity assays were conducted with 25 μ m [3H]AdoMet and 500 μ m histone H3 peptide (residues 1–20) with varying concentrations of WRAD ( open triangles ) or WRA ( open circles ). Each point corresponds to the average of duplicate measurements with the error bars indicating the standard error of measurement. Linear regression fitting of the data gave slopes of 0.0016 and 0.001, and R 2 values of 0.99 and 0.97 for WRAD and WRA complexes, respectively. C , diffusion-free sedimentation coefficient distributions ( c ( s )) derived from sedimentation velocity analytical ultracentrifugation of WRAD ( upper panel ) and WRA ( lower panel ) complexes at concentrations of 2.2 μ m ( solid line ), 1.1 μ m ( dashed line ), and 0.55 μ m ( dotted line ).

    Journal:

    Article Title: A Novel Non-SET Domain Multi-subunit Methyltransferase Required for Sequential Nucleosomal Histone H3 Methylation by the Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex

    doi: 10.1074/jbc.M110.174524

    Figure Lengend Snippet: WDR5, RbBP5, and Ash2L are required for the H3K4 methylation activity of WRAD. A , comparison of enzymatic activity of individual WRAD components and all possible binary, ternary, and quaternary complexes. W , WDR5; R , RbBP5; A , Ash2L; D , DPY-30. Histone methyltransferase assays were conducted for a period of 8 h using [3 H]AdoMet and chicken core histones as the substrate. Quenched reactions were separated by 18% Tris-glycine SDS-PAGE and visualized with Coomassie Brilliant Blue ( upper panels ) and fluorography ( lower panels ). A no-enzyme control is shown in lane 16. B , WRAD and WRA histone methyltransferase activity as a function of enzyme concentration. Methyltransferase activity assays were conducted with 25 μ m [3H]AdoMet and 500 μ m histone H3 peptide (residues 1–20) with varying concentrations of WRAD ( open triangles ) or WRA ( open circles ). Each point corresponds to the average of duplicate measurements with the error bars indicating the standard error of measurement. Linear regression fitting of the data gave slopes of 0.0016 and 0.001, and R 2 values of 0.99 and 0.97 for WRAD and WRA complexes, respectively. C , diffusion-free sedimentation coefficient distributions ( c ( s )) derived from sedimentation velocity analytical ultracentrifugation of WRAD ( upper panel ) and WRA ( lower panel ) complexes at concentrations of 2.2 μ m ( solid line ), 1.1 μ m ( dashed line ), and 0.55 μ m ( dotted line ).

    Article Snippet: Enzymatic reactions (20 μl) were initiated by the addition of enzyme (1–17 μ m ) to reaction mixtures containing a fixed concentration of histone H3 peptide (residues 1–20) and variable concentrations of [3 H]AdoMet (PerkinElmer Life Sciences; specific activity, 12 μCi/m m ) or fixed [3 H]AdoMet concentrations and variable concentrations of histone H3 peptide in assay buffer (50 m m Tris, pH 8.5, 200 m m NaCl, 3 m m DTT, 5 m m MgCl2 , and 5% glycerol).

    Techniques: Methylation, Activity Assay, SDS Page, Concentration Assay, Diffusion-based Assay, Sedimentation, Derivative Assay

    WRAD is a histone H3 lysine 4-specific monomethyltransferase. A , enzymatic assays showing the methyltransferase activity of WRAD after a period of 8 h using [3 H]methyl- S -adenosylmethionine and purified chicken core histones as substrates. Enzymatic reactions were separated by 18% Tris-glycine SDS-PAGE and visualized with Coomassie Brilliant Blue ( left panel , lanes 1 and 2 ) or fluorography ( right panel , lanes 3 and 4 ). B , enzymatic activity of WRAD using 4 μg of full-length histone H3 proteins (Active Motif®) that were either unmodified ( H3 , lane 1 ) or previously monomethylated at lysine 4 ( H3K4me1 , lane 2 ). The reactions were separated by 18% Tris-glycine PAGE and visualized with Coomassie Brilliant Blue ( upper panel ) and fluorography (3 H-Methyl , lower panel ). C , enzymatic activity of MLL1 core complex after a period of 8 h using 4 μg of full-length histone H3 proteins that were either unmodified ( H3 , lane 1 ) or previously monomethylated at lysine 4 ( H3K4me1 , lane 2 ). The reaction products were separated by 4–12% Bis-Tris PAGE and visualized as described in B. D , comparison of the enzymatic activity of WRAD using as substrates recombinant histone H3 (1.5 μg, lane 1 ), dialyzed histone octamer (6 μg, lane 2 ), or an equivalent amount of reconstituted nucleosomes ( lane 3 ). The reactions were separated by 18% Tris-glycine SDS-PAGE and visualized as described in B above. Octamer * denotes that the histone octamer dissociates into one H3/H4 tetramer and two H2A/H2B dimers upon dialysis into assay buffer (see text).

    Journal:

    Article Title: A Novel Non-SET Domain Multi-subunit Methyltransferase Required for Sequential Nucleosomal Histone H3 Methylation by the Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex

    doi: 10.1074/jbc.M110.174524

    Figure Lengend Snippet: WRAD is a histone H3 lysine 4-specific monomethyltransferase. A , enzymatic assays showing the methyltransferase activity of WRAD after a period of 8 h using [3 H]methyl- S -adenosylmethionine and purified chicken core histones as substrates. Enzymatic reactions were separated by 18% Tris-glycine SDS-PAGE and visualized with Coomassie Brilliant Blue ( left panel , lanes 1 and 2 ) or fluorography ( right panel , lanes 3 and 4 ). B , enzymatic activity of WRAD using 4 μg of full-length histone H3 proteins (Active Motif®) that were either unmodified ( H3 , lane 1 ) or previously monomethylated at lysine 4 ( H3K4me1 , lane 2 ). The reactions were separated by 18% Tris-glycine PAGE and visualized with Coomassie Brilliant Blue ( upper panel ) and fluorography (3 H-Methyl , lower panel ). C , enzymatic activity of MLL1 core complex after a period of 8 h using 4 μg of full-length histone H3 proteins that were either unmodified ( H3 , lane 1 ) or previously monomethylated at lysine 4 ( H3K4me1 , lane 2 ). The reaction products were separated by 4–12% Bis-Tris PAGE and visualized as described in B. D , comparison of the enzymatic activity of WRAD using as substrates recombinant histone H3 (1.5 μg, lane 1 ), dialyzed histone octamer (6 μg, lane 2 ), or an equivalent amount of reconstituted nucleosomes ( lane 3 ). The reactions were separated by 18% Tris-glycine SDS-PAGE and visualized as described in B above. Octamer * denotes that the histone octamer dissociates into one H3/H4 tetramer and two H2A/H2B dimers upon dialysis into assay buffer (see text).

    Article Snippet: Enzymatic reactions (20 μl) were initiated by the addition of enzyme (1–17 μ m ) to reaction mixtures containing a fixed concentration of histone H3 peptide (residues 1–20) and variable concentrations of [3 H]AdoMet (PerkinElmer Life Sciences; specific activity, 12 μCi/m m ) or fixed [3 H]AdoMet concentrations and variable concentrations of histone H3 peptide in assay buffer (50 m m Tris, pH 8.5, 200 m m NaCl, 3 m m DTT, 5 m m MgCl2 , and 5% glycerol).

    Techniques: Activity Assay, Purification, SDS Page, Polyacrylamide Gel Electrophoresis, Recombinant

    Steady-state kinetics and inhibition analysis of WRA(D). A , comparison of WRA ( open circle ) and WRAD ( open triangle ) kinetics with AdoMet as the variable substrate (ranging from 0.5 to 25 μ m ) and fixed concentrations (1 m m ) of histone H3 peptide (residues 1–20). Rates of methylation are the means of duplicate measurements ± standard error of measurement. Apparent kinetic parameters were determined by fitting the data to the Michaelis-Menten equation ( , “Experimental Procedures”). B , comparison of WRA ( open circles ) and WRAD ( open triangles ) kinetics with histone H3 peptide as the variable substrate (ranging from 25 to 5000 μ m ). The data are represented and fitted as described for A . For clarity, the values for the concentration range from 0 to 1000 μ m are shown. C , comparison of the enzymatic activity of WRAD (4.3 μ m ) with increasing concentrations of AdoHyc (1–250 μ m ). Activity assays were conducted with fixed concentrations of AdoMet (25 μ m ) and histone H3 peptide (500 μ m ). Each point represents the means ± S.E. of measurement from duplicate measurements. The data were fit to (“Experimental Procedures”).

    Journal:

    Article Title: A Novel Non-SET Domain Multi-subunit Methyltransferase Required for Sequential Nucleosomal Histone H3 Methylation by the Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex

    doi: 10.1074/jbc.M110.174524

    Figure Lengend Snippet: Steady-state kinetics and inhibition analysis of WRA(D). A , comparison of WRA ( open circle ) and WRAD ( open triangle ) kinetics with AdoMet as the variable substrate (ranging from 0.5 to 25 μ m ) and fixed concentrations (1 m m ) of histone H3 peptide (residues 1–20). Rates of methylation are the means of duplicate measurements ± standard error of measurement. Apparent kinetic parameters were determined by fitting the data to the Michaelis-Menten equation ( , “Experimental Procedures”). B , comparison of WRA ( open circles ) and WRAD ( open triangles ) kinetics with histone H3 peptide as the variable substrate (ranging from 25 to 5000 μ m ). The data are represented and fitted as described for A . For clarity, the values for the concentration range from 0 to 1000 μ m are shown. C , comparison of the enzymatic activity of WRAD (4.3 μ m ) with increasing concentrations of AdoHyc (1–250 μ m ). Activity assays were conducted with fixed concentrations of AdoMet (25 μ m ) and histone H3 peptide (500 μ m ). Each point represents the means ± S.E. of measurement from duplicate measurements. The data were fit to (“Experimental Procedures”).

    Article Snippet: Enzymatic reactions (20 μl) were initiated by the addition of enzyme (1–17 μ m ) to reaction mixtures containing a fixed concentration of histone H3 peptide (residues 1–20) and variable concentrations of [3 H]AdoMet (PerkinElmer Life Sciences; specific activity, 12 μCi/m m ) or fixed [3 H]AdoMet concentrations and variable concentrations of histone H3 peptide in assay buffer (50 m m Tris, pH 8.5, 200 m m NaCl, 3 m m DTT, 5 m m MgCl2 , and 5% glycerol).

    Techniques: Inhibition, Methylation, Concentration Assay, Activity Assay