s adenosylmethionine  (New England Biolabs)


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    S adenosylmethionine SAM 32mM
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    S adenosylmethionine SAM 32mM 0 5 ml
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    New England Biolabs s adenosylmethionine
    S adenosylmethionine SAM 32mM
    S adenosylmethionine SAM 32mM 0 5 ml
    https://www.bioz.com/result/s adenosylmethionine/product/New England Biolabs
    Average 99 stars, based on 134 article reviews
    Price from $9.99 to $1999.99
    s adenosylmethionine - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿"

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00108-07

    Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.
    Figure Legend Snippet: Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Techniques Used: Purification, Incubation, Polymerase Chain Reaction, Concentration Assay, In Vitro

    2) Product Images from "Convergent evolution between PALI1 and JARID2 for the allosteric activation of PRC2"

    Article Title: Convergent evolution between PALI1 and JARID2 for the allosteric activation of PRC2

    Journal: bioRxiv

    doi: 10.1101/2020.05.28.122556

    PALI1 is methylated in vitro and in vivo . a, Schematic representation of the PRC2 methylome in vivo and in vitro , as identified from MS/MS data. Mouse and human icon represent the organism of origin and cell lines are indicated (see methods section for references and accession numbers of the raw MS/MS data). Test tube icons representing methylations in the purified recombinant human PRC2-PALI1 PIR-long . PALI truncations used in this study are indicated in purple (upper right). Residues are indicated with asterisks where the position probability of the methylation is less than 0.95 (see Supplementary Table 1 for the values). PRC2.1 and PRC2.2 accessory subunits are in blue and red, respectively, and core subunits are in grey. b, Coomassie blue-stained SDS-PAGE of recombinant human PRC2-PALI1 PIR complexes, as indicated. c, HTMase assay of the PRC2-[MBP-PALI1 PIR ] complex using mononucleosomes substrate were carried out in the presence or absence of C3 protease to confirm that PALI1 PIR is methylated. The MBP-cleaved and uncleaved PALI1 PIR bands are indicated on the radiogram with asterisks.
    Figure Legend Snippet: PALI1 is methylated in vitro and in vivo . a, Schematic representation of the PRC2 methylome in vivo and in vitro , as identified from MS/MS data. Mouse and human icon represent the organism of origin and cell lines are indicated (see methods section for references and accession numbers of the raw MS/MS data). Test tube icons representing methylations in the purified recombinant human PRC2-PALI1 PIR-long . PALI truncations used in this study are indicated in purple (upper right). Residues are indicated with asterisks where the position probability of the methylation is less than 0.95 (see Supplementary Table 1 for the values). PRC2.1 and PRC2.2 accessory subunits are in blue and red, respectively, and core subunits are in grey. b, Coomassie blue-stained SDS-PAGE of recombinant human PRC2-PALI1 PIR complexes, as indicated. c, HTMase assay of the PRC2-[MBP-PALI1 PIR ] complex using mononucleosomes substrate were carried out in the presence or absence of C3 protease to confirm that PALI1 PIR is methylated. The MBP-cleaved and uncleaved PALI1 PIR bands are indicated on the radiogram with asterisks.

    Techniques Used: Methylation, In Vitro, In Vivo, Tandem Mass Spectroscopy, Purification, Recombinant, Staining, SDS Page

    3) Product Images from "A post‐translational modification switch controls coactivator function of histone methyltransferases G9a and GLP"

    Article Title: A post‐translational modification switch controls coactivator function of histone methyltransferases G9a and GLP

    Journal: EMBO Reports

    doi: 10.15252/embr.201744060

    G9a and GLP are methylated on their N‐terminal domain in cells Schematic representation of the related proteins GLP (EHMT1) and G9a (EHMT2). N: N‐terminal coactivator domain, E: polyglutamate domain, Cys: cysteine‐rich region, ANK: six ankyrin repeats, SET: SET‐domain containing methyltransferase activity. Partial protein sequence of hG9a and hGLP homologs shows the hypothetical methylated lysine residues (K) in red. After protein methylation reactions, in vitro methylated proteins were detected by immunoblot with pan‐methyllysine antibody (pan met‐K). The corresponding Coomassie‐stained gels are shown as loading controls. SAM, S‐adenosylmethionine. Cos‐7 cells were transfected with plasmids encoding full‐length HA‐hG9a wild type or K185R mutant, or full‐length HA‐hGLP wild type or K205R mutant. Lysates were immunoprecipitated (IP) with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Expression of HA‐tagged proteins and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input). Cos‐7 cells were transfected with a plasmid encoding full‐length HA‐hG9a and treated with 2 μM UNC0646 or vehicle DMSO for 24 h. Lysates were immunoprecipitated with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Methylation and phosphorylation of endogenous G9a and GLP in A549 cells treated with 100 nM dex for 4 h were analyzed by immunoprecipitation with control IgG antibody, anti‐G9a (top), or anti‐GLP (bottom), followed by immunoblot with antibodies listed. Expression of G9a, GLP, and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input).
    Figure Legend Snippet: G9a and GLP are methylated on their N‐terminal domain in cells Schematic representation of the related proteins GLP (EHMT1) and G9a (EHMT2). N: N‐terminal coactivator domain, E: polyglutamate domain, Cys: cysteine‐rich region, ANK: six ankyrin repeats, SET: SET‐domain containing methyltransferase activity. Partial protein sequence of hG9a and hGLP homologs shows the hypothetical methylated lysine residues (K) in red. After protein methylation reactions, in vitro methylated proteins were detected by immunoblot with pan‐methyllysine antibody (pan met‐K). The corresponding Coomassie‐stained gels are shown as loading controls. SAM, S‐adenosylmethionine. Cos‐7 cells were transfected with plasmids encoding full‐length HA‐hG9a wild type or K185R mutant, or full‐length HA‐hGLP wild type or K205R mutant. Lysates were immunoprecipitated (IP) with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Expression of HA‐tagged proteins and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input). Cos‐7 cells were transfected with a plasmid encoding full‐length HA‐hG9a and treated with 2 μM UNC0646 or vehicle DMSO for 24 h. Lysates were immunoprecipitated with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Methylation and phosphorylation of endogenous G9a and GLP in A549 cells treated with 100 nM dex for 4 h were analyzed by immunoprecipitation with control IgG antibody, anti‐G9a (top), or anti‐GLP (bottom), followed by immunoblot with antibodies listed. Expression of G9a, GLP, and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input).

    Techniques Used: Methylation, Activity Assay, Sequencing, In Vitro, Staining, Transfection, Mutagenesis, Immunoprecipitation, Expressing, Plasmid Preparation

    4) Product Images from "Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF"

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp524

    ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.
    Figure Legend Snippet: ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Techniques Used: Electrophoresis, Incubation, Methylation, Nucleic Acid Electrophoresis, Plasmid Preparation, Staining, Amplification, Polymerase Chain Reaction, Clone Assay, Generated, Concentration Assay

    5) Product Images from "RNA matchmaking remodels lncRNA structure and promotes PRC2 activity"

    Article Title: RNA matchmaking remodels lncRNA structure and promotes PRC2 activity

    Journal: bioRxiv

    doi: 10.1101/2020.04.13.040071

    Duplex RNA promotes PRC2 activity. a , Native gel of di-nucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40-bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. b , Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4 and AEBP2, analyzed by SDS-PAGE and stained with Coomassie blue. c , Histone methyltransferase assay (HMTase assay) was performed with recombinant PRC2 complex, di-nucleosomes, S-Adenosylmethionine (SAM) with and without the co-factor JARID2 (amino acids 119-574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. d , Native 0.5X TBE gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration.
    Figure Legend Snippet: Duplex RNA promotes PRC2 activity. a , Native gel of di-nucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40-bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. b , Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4 and AEBP2, analyzed by SDS-PAGE and stained with Coomassie blue. c , Histone methyltransferase assay (HMTase assay) was performed with recombinant PRC2 complex, di-nucleosomes, S-Adenosylmethionine (SAM) with and without the co-factor JARID2 (amino acids 119-574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. d , Native 0.5X TBE gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration.

    Techniques Used: Activity Assay, Staining, Recombinant, SDS Page, HMT Assay, Western Blot, Titration

    6) Product Images from "Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancer growth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications"

    Article Title: Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancer growth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23704

    Effect of S-adenosylmethionine (SAM) on breast cancer cell proliferation, migration, invasion, anchorage-independent growth, and apoptosis in vitro (A) Schematic diagram of the treatment strategy for all the in vitro experiments. Human breast cancer cells MDA-MB-231 and Hs578T were treated with SAM (100 and 200 μM) by directly adding it to regular growth medium every other day from day 2 until they were harvested. (B) Human breast cancer cells MDA-MB-231 and Hs578T were plated in 6-well plates and treated with vehicle alone as control or SAM (100 and 200 μM). Cell growth rate in each group was determined on day 1, 3, 5, and 7 by Coulter counter as described in Methods. Results are shown as bar graphs of data obtained from three different experiments. (C) Wound healing assay for determining the migration capacity of the cells was carried out by making a cross-like scratch on the plate when they reached 90% confluency. Control and SAM (100 and 200 μM) treated cells were grown in culture media containing 2% FBS and migrating cells were photographed and recorded at different time points, and percentage of wound healing with respect to initial scratch (T0) was calculated using the equation described in ‘Supplementary Materials’. The results are represented as bar graphs obtained from three experiments. (D) Boyden chamber Matrigel invasion assay was used to measure the invasiveness of control and SAM-treated (100 and 200 μM) MDA-MB-231 and Hs578T cells. The cells were placed in the upper chamber, and conditioned media used as ‘chemoattractant’ was added into the lower chamber. Following an incubation period of 18 hours, the invasion process was stopped and the invaded cells from control and 100 and 200 μM SAM-treated groups were fixed, stained and randomly selected fields were counted under the microscope and averaged. Representative image of one randomly selected field for each treatment for both cell lines along with the number of cells invaded per field are shown. (E) After the usual treatment regimen, 5 × 10 3 cell from control and SAM-treated (100 μM and 200 μM) groups were plated onto soft agar for anchorage-independent growth assay. The culture media was replenished every other day for two weeks, and the number of colonies was counted. (F) Apoptosis was determined by flow cytometry after staining the control and SAM-treated cells with Annexin V/propidium iodide. Representative contour plots of annexinV-FITC staining of apoptotic cells vs. PI staining for both control and SAM-treated (100 μM) cells are shown. The bar graphs on the right panels show the total percentages of apoptotic cells for different treatments. Results are presented as the mean ± SEM from control and SAM-treated experimental cells. Significant differences were determined using ANOVA followed by post hoc Bonferroni test and are represented by asterisks ( * P
    Figure Legend Snippet: Effect of S-adenosylmethionine (SAM) on breast cancer cell proliferation, migration, invasion, anchorage-independent growth, and apoptosis in vitro (A) Schematic diagram of the treatment strategy for all the in vitro experiments. Human breast cancer cells MDA-MB-231 and Hs578T were treated with SAM (100 and 200 μM) by directly adding it to regular growth medium every other day from day 2 until they were harvested. (B) Human breast cancer cells MDA-MB-231 and Hs578T were plated in 6-well plates and treated with vehicle alone as control or SAM (100 and 200 μM). Cell growth rate in each group was determined on day 1, 3, 5, and 7 by Coulter counter as described in Methods. Results are shown as bar graphs of data obtained from three different experiments. (C) Wound healing assay for determining the migration capacity of the cells was carried out by making a cross-like scratch on the plate when they reached 90% confluency. Control and SAM (100 and 200 μM) treated cells were grown in culture media containing 2% FBS and migrating cells were photographed and recorded at different time points, and percentage of wound healing with respect to initial scratch (T0) was calculated using the equation described in ‘Supplementary Materials’. The results are represented as bar graphs obtained from three experiments. (D) Boyden chamber Matrigel invasion assay was used to measure the invasiveness of control and SAM-treated (100 and 200 μM) MDA-MB-231 and Hs578T cells. The cells were placed in the upper chamber, and conditioned media used as ‘chemoattractant’ was added into the lower chamber. Following an incubation period of 18 hours, the invasion process was stopped and the invaded cells from control and 100 and 200 μM SAM-treated groups were fixed, stained and randomly selected fields were counted under the microscope and averaged. Representative image of one randomly selected field for each treatment for both cell lines along with the number of cells invaded per field are shown. (E) After the usual treatment regimen, 5 × 10 3 cell from control and SAM-treated (100 μM and 200 μM) groups were plated onto soft agar for anchorage-independent growth assay. The culture media was replenished every other day for two weeks, and the number of colonies was counted. (F) Apoptosis was determined by flow cytometry after staining the control and SAM-treated cells with Annexin V/propidium iodide. Representative contour plots of annexinV-FITC staining of apoptotic cells vs. PI staining for both control and SAM-treated (100 μM) cells are shown. The bar graphs on the right panels show the total percentages of apoptotic cells for different treatments. Results are presented as the mean ± SEM from control and SAM-treated experimental cells. Significant differences were determined using ANOVA followed by post hoc Bonferroni test and are represented by asterisks ( * P

    Techniques Used: Migration, In Vitro, Multiple Displacement Amplification, Wound Healing Assay, Invasion Assay, Incubation, Staining, Microscopy, Growth Assay, Flow Cytometry, Cytometry

    Related Articles

    Amplification:

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿
    Article Snippet: .. To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min. ..

    In Vitro:

    Article Title: Mechanisms of Differential Expression of the CYP2A13 7520C and 7520G Alleles in Human Lung: Allelic Expression Analysis for CYP2A13 Heterogeneous Nuclear RNA, and Evidence for the Involvement of Multiple Cis-Regulatory Single Nucleotide Polymorphisms
    Article Snippet: .. The p2A13_−1479T plasmid, containing a 2-kb CYP2A13 * 1 promoter fragment in a pGL3_Basic (Promega, Madison, WI) luciferase reporter vector, was originally named p2A13_2k [ ]; the p2A13_−1479C plasmid contains a C at −1479, instead of a T. The p2A13_−1479C and p2A13_−1479T constructs were methylated in vitro with CpG methylase M. SssI (New England Biolabs, Ipswich, MA), in the presence of 16 μmol/ L S-adenosylmethionine (New England Biolabs). .. The reaction was carried out at 37°C for 6 h. Complete methylation was confirmed by digestion with methylation-sensitive BstU I (New England Biolabs).

    Methylation:

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿
    Article Snippet: .. To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min. ..

    Article Title: Alterations of sorbin and SH3 domain containing 3 (SORBS3) in human skeletal muscle following Roux-en-Y gastric bypass surgery
    Article Snippet: .. The SORBS3 construct was either mock methylated or methylated using 1600 μM S-adenosylmethionine (SAM) and two different DNA methyltransferases, SssI and HhaI (New England Biolabs, Frankfurt, Germany). .. Mouse muscle cell lines C2C12 were cultured in DMEM, supplemented with 10% serum and 1% of an antibiotic/antimycotic mixture.

    Article Title: Mechanisms of Differential Expression of the CYP2A13 7520C and 7520G Alleles in Human Lung: Allelic Expression Analysis for CYP2A13 Heterogeneous Nuclear RNA, and Evidence for the Involvement of Multiple Cis-Regulatory Single Nucleotide Polymorphisms
    Article Snippet: .. The p2A13_−1479T plasmid, containing a 2-kb CYP2A13 * 1 promoter fragment in a pGL3_Basic (Promega, Madison, WI) luciferase reporter vector, was originally named p2A13_2k [ ]; the p2A13_−1479C plasmid contains a C at −1479, instead of a T. The p2A13_−1479C and p2A13_−1479T constructs were methylated in vitro with CpG methylase M. SssI (New England Biolabs, Ipswich, MA), in the presence of 16 μmol/ L S-adenosylmethionine (New England Biolabs). .. The reaction was carried out at 37°C for 6 h. Complete methylation was confirmed by digestion with methylation-sensitive BstU I (New England Biolabs).

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF
    Article Snippet: .. Reaction were stopped by addition of 10 mM ADP and incubation on ice for 10 min. Methylation of the remodeled products was performed by incubation at 37°C for 15 min after addition of 5 U M.SssI and S-adenosylmethionine to a final concentration of 160 μM (New England BioLabs). .. After addition of competitor plasmid DNA, samples were further incubated on ice for 10 min and separated by native gel electrophoresis for about 2 h and stained with ethidium bromide for 10 min. Major bands were excised and eluted from the gel by overnight incubation in 400 μl of 10 mM Tris pH 8.0, 1 mM EDTA at 55°C.

    Mutagenesis:

    Article Title: A post‐translational modification switch controls coactivator function of histone methyltransferases G9a and GLP
    Article Snippet: .. Bacterially produced GST fusion proteins (2 μg) of N‐terminal fragments of G9a or GLP (GST‐hG9a N or GST‐hGLP N), mutant (GST‐hG9a N K185R or GST‐hGLP N K205R or GST‐hG9a N T186A), or GST alone were incubated 90 min at 30°C with GST‐hG9a ΔN or GST‐hGLP ΔN in the presence (or not) of 1 mM of unradiolabeled SAM (New England Biolabs, B9003S). .. Methylated products were analyzed by standard SDS gel electrophoresis followed by immunoblot.

    Construct:

    Article Title: Alterations of sorbin and SH3 domain containing 3 (SORBS3) in human skeletal muscle following Roux-en-Y gastric bypass surgery
    Article Snippet: .. The SORBS3 construct was either mock methylated or methylated using 1600 μM S-adenosylmethionine (SAM) and two different DNA methyltransferases, SssI and HhaI (New England Biolabs, Frankfurt, Germany). .. Mouse muscle cell lines C2C12 were cultured in DMEM, supplemented with 10% serum and 1% of an antibiotic/antimycotic mixture.

    Article Title: Mechanisms of Differential Expression of the CYP2A13 7520C and 7520G Alleles in Human Lung: Allelic Expression Analysis for CYP2A13 Heterogeneous Nuclear RNA, and Evidence for the Involvement of Multiple Cis-Regulatory Single Nucleotide Polymorphisms
    Article Snippet: .. The p2A13_−1479T plasmid, containing a 2-kb CYP2A13 * 1 promoter fragment in a pGL3_Basic (Promega, Madison, WI) luciferase reporter vector, was originally named p2A13_2k [ ]; the p2A13_−1479C plasmid contains a C at −1479, instead of a T. The p2A13_−1479C and p2A13_−1479T constructs were methylated in vitro with CpG methylase M. SssI (New England Biolabs, Ipswich, MA), in the presence of 16 μmol/ L S-adenosylmethionine (New England Biolabs). .. The reaction was carried out at 37°C for 6 h. Complete methylation was confirmed by digestion with methylation-sensitive BstU I (New England Biolabs).

    Produced:

    Article Title: A post‐translational modification switch controls coactivator function of histone methyltransferases G9a and GLP
    Article Snippet: .. Bacterially produced GST fusion proteins (2 μg) of N‐terminal fragments of G9a or GLP (GST‐hG9a N or GST‐hGLP N), mutant (GST‐hG9a N K185R or GST‐hGLP N K205R or GST‐hG9a N T186A), or GST alone were incubated 90 min at 30°C with GST‐hG9a ΔN or GST‐hGLP ΔN in the presence (or not) of 1 mM of unradiolabeled SAM (New England Biolabs, B9003S). .. Methylated products were analyzed by standard SDS gel electrophoresis followed by immunoblot.

    Concentration Assay:

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF
    Article Snippet: .. Reaction were stopped by addition of 10 mM ADP and incubation on ice for 10 min. Methylation of the remodeled products was performed by incubation at 37°C for 15 min after addition of 5 U M.SssI and S-adenosylmethionine to a final concentration of 160 μM (New England BioLabs). .. After addition of competitor plasmid DNA, samples were further incubated on ice for 10 min and separated by native gel electrophoresis for about 2 h and stained with ethidium bromide for 10 min. Major bands were excised and eluted from the gel by overnight incubation in 400 μl of 10 mM Tris pH 8.0, 1 mM EDTA at 55°C.

    Incubation:

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿
    Article Snippet: .. To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min. ..

    Article Title: A post‐translational modification switch controls coactivator function of histone methyltransferases G9a and GLP
    Article Snippet: .. Bacterially produced GST fusion proteins (2 μg) of N‐terminal fragments of G9a or GLP (GST‐hG9a N or GST‐hGLP N), mutant (GST‐hG9a N K185R or GST‐hGLP N K205R or GST‐hG9a N T186A), or GST alone were incubated 90 min at 30°C with GST‐hG9a ΔN or GST‐hGLP ΔN in the presence (or not) of 1 mM of unradiolabeled SAM (New England Biolabs, B9003S). .. Methylated products were analyzed by standard SDS gel electrophoresis followed by immunoblot.

    Article Title: Epigenetic Regulation of PLIN1 in Obese Women and its Relation to Lipolysis
    Article Snippet: .. In brief, 10–15 µg of plasmid DNA was incubated with or without Sss I methyltransferase (20 U/µl; 2 U/µg DNA) in the presence of 640 µM S-Adenosylmethionine (SAM) (New England Biolabs) for four hours at 37 °C, with another 640 µM SAM being added after the first two hours of incubation. .. Plasmid DNA was purified using QIAquick PCR purification kit (Qiagen).

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF
    Article Snippet: .. Reaction were stopped by addition of 10 mM ADP and incubation on ice for 10 min. Methylation of the remodeled products was performed by incubation at 37°C for 15 min after addition of 5 U M.SssI and S-adenosylmethionine to a final concentration of 160 μM (New England BioLabs). .. After addition of competitor plasmid DNA, samples were further incubated on ice for 10 min and separated by native gel electrophoresis for about 2 h and stained with ethidium bromide for 10 min. Major bands were excised and eluted from the gel by overnight incubation in 400 μl of 10 mM Tris pH 8.0, 1 mM EDTA at 55°C.

    Luciferase:

    Article Title: Mechanisms of Differential Expression of the CYP2A13 7520C and 7520G Alleles in Human Lung: Allelic Expression Analysis for CYP2A13 Heterogeneous Nuclear RNA, and Evidence for the Involvement of Multiple Cis-Regulatory Single Nucleotide Polymorphisms
    Article Snippet: .. The p2A13_−1479T plasmid, containing a 2-kb CYP2A13 * 1 promoter fragment in a pGL3_Basic (Promega, Madison, WI) luciferase reporter vector, was originally named p2A13_2k [ ]; the p2A13_−1479C plasmid contains a C at −1479, instead of a T. The p2A13_−1479C and p2A13_−1479T constructs were methylated in vitro with CpG methylase M. SssI (New England Biolabs, Ipswich, MA), in the presence of 16 μmol/ L S-adenosylmethionine (New England Biolabs). .. The reaction was carried out at 37°C for 6 h. Complete methylation was confirmed by digestion with methylation-sensitive BstU I (New England Biolabs).

    Polymerase Chain Reaction:

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿
    Article Snippet: .. To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min. ..

    Recombinant:

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿
    Article Snippet: .. To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min. ..

    Article Title: RNA matchmaking remodels lncRNA structure and promotes PRC2 activity
    Article Snippet: .. Histone methyltransferase assays HMTase assays were performed in a total volume of 15 μl containing HMTase buffer (10 mM HEPES, pH 7.5, 2.5 mM MgCl2 , 0.25 mM EDTA, 4% Glycerol and 0.1 mM DTT) with 75 μM S-Adenosylmethionine (SAM, NEB), varying amounts ssRNA and duplex RNA (see above), 600 nM JARID2, 360 nM of dinucleosomes, and 600-660 nM recombinant human PRC2 complexes under the following conditions. .. The reaction mixture was incubated for 30 minutes at 30°C and stopped by adding 12 μl of Laemmli sample buffer (Biorad).

    Plasmid Preparation:

    Article Title: Mechanisms of Differential Expression of the CYP2A13 7520C and 7520G Alleles in Human Lung: Allelic Expression Analysis for CYP2A13 Heterogeneous Nuclear RNA, and Evidence for the Involvement of Multiple Cis-Regulatory Single Nucleotide Polymorphisms
    Article Snippet: .. The p2A13_−1479T plasmid, containing a 2-kb CYP2A13 * 1 promoter fragment in a pGL3_Basic (Promega, Madison, WI) luciferase reporter vector, was originally named p2A13_2k [ ]; the p2A13_−1479C plasmid contains a C at −1479, instead of a T. The p2A13_−1479C and p2A13_−1479T constructs were methylated in vitro with CpG methylase M. SssI (New England Biolabs, Ipswich, MA), in the presence of 16 μmol/ L S-adenosylmethionine (New England Biolabs). .. The reaction was carried out at 37°C for 6 h. Complete methylation was confirmed by digestion with methylation-sensitive BstU I (New England Biolabs).

    Article Title: Epigenetic Regulation of PLIN1 in Obese Women and its Relation to Lipolysis
    Article Snippet: .. In brief, 10–15 µg of plasmid DNA was incubated with or without Sss I methyltransferase (20 U/µl; 2 U/µg DNA) in the presence of 640 µM S-Adenosylmethionine (SAM) (New England Biolabs) for four hours at 37 °C, with another 640 µM SAM being added after the first two hours of incubation. .. Plasmid DNA was purified using QIAquick PCR purification kit (Qiagen).

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    New England Biolabs s adenosylmethionine
    Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S <t>-adenosylmethionine.</t> HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.
    S Adenosylmethionine, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Journal: Journal of Bacteriology

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿

    doi: 10.1128/JB.00108-07

    Figure Lengend Snippet: Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Article Snippet: To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min.

    Techniques: Purification, Incubation, Polymerase Chain Reaction, Concentration Assay, In Vitro

    G9a and GLP are methylated on their N‐terminal domain in cells Schematic representation of the related proteins GLP (EHMT1) and G9a (EHMT2). N: N‐terminal coactivator domain, E: polyglutamate domain, Cys: cysteine‐rich region, ANK: six ankyrin repeats, SET: SET‐domain containing methyltransferase activity. Partial protein sequence of hG9a and hGLP homologs shows the hypothetical methylated lysine residues (K) in red. After protein methylation reactions, in vitro methylated proteins were detected by immunoblot with pan‐methyllysine antibody (pan met‐K). The corresponding Coomassie‐stained gels are shown as loading controls. SAM, S‐adenosylmethionine. Cos‐7 cells were transfected with plasmids encoding full‐length HA‐hG9a wild type or K185R mutant, or full‐length HA‐hGLP wild type or K205R mutant. Lysates were immunoprecipitated (IP) with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Expression of HA‐tagged proteins and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input). Cos‐7 cells were transfected with a plasmid encoding full‐length HA‐hG9a and treated with 2 μM UNC0646 or vehicle DMSO for 24 h. Lysates were immunoprecipitated with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Methylation and phosphorylation of endogenous G9a and GLP in A549 cells treated with 100 nM dex for 4 h were analyzed by immunoprecipitation with control IgG antibody, anti‐G9a (top), or anti‐GLP (bottom), followed by immunoblot with antibodies listed. Expression of G9a, GLP, and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input).

    Journal: EMBO Reports

    Article Title: A post‐translational modification switch controls coactivator function of histone methyltransferases G9a and GLP

    doi: 10.15252/embr.201744060

    Figure Lengend Snippet: G9a and GLP are methylated on their N‐terminal domain in cells Schematic representation of the related proteins GLP (EHMT1) and G9a (EHMT2). N: N‐terminal coactivator domain, E: polyglutamate domain, Cys: cysteine‐rich region, ANK: six ankyrin repeats, SET: SET‐domain containing methyltransferase activity. Partial protein sequence of hG9a and hGLP homologs shows the hypothetical methylated lysine residues (K) in red. After protein methylation reactions, in vitro methylated proteins were detected by immunoblot with pan‐methyllysine antibody (pan met‐K). The corresponding Coomassie‐stained gels are shown as loading controls. SAM, S‐adenosylmethionine. Cos‐7 cells were transfected with plasmids encoding full‐length HA‐hG9a wild type or K185R mutant, or full‐length HA‐hGLP wild type or K205R mutant. Lysates were immunoprecipitated (IP) with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Expression of HA‐tagged proteins and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input). Cos‐7 cells were transfected with a plasmid encoding full‐length HA‐hG9a and treated with 2 μM UNC0646 or vehicle DMSO for 24 h. Lysates were immunoprecipitated with pan met‐K antibody and immunoblotted with HA antibody (top), or the usage of the two antibodies was reversed (bottom). Methylation and phosphorylation of endogenous G9a and GLP in A549 cells treated with 100 nM dex for 4 h were analyzed by immunoprecipitation with control IgG antibody, anti‐G9a (top), or anti‐GLP (bottom), followed by immunoblot with antibodies listed. Expression of G9a, GLP, and β‐actin (loading control) in the unfractionated extracts is shown at the bottom (Input).

    Article Snippet: Bacterially produced GST fusion proteins (2 μg) of N‐terminal fragments of G9a or GLP (GST‐hG9a N or GST‐hGLP N), mutant (GST‐hG9a N K185R or GST‐hGLP N K205R or GST‐hG9a N T186A), or GST alone were incubated 90 min at 30°C with GST‐hG9a ΔN or GST‐hGLP ΔN in the presence (or not) of 1 mM of unradiolabeled SAM (New England Biolabs, B9003S).

    Techniques: Methylation, Activity Assay, Sequencing, In Vitro, Staining, Transfection, Mutagenesis, Immunoprecipitation, Expressing, Plasmid Preparation

    ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Journal: Nucleic Acids Research

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    doi: 10.1093/nar/gkp524

    Figure Lengend Snippet: ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Article Snippet: Reaction were stopped by addition of 10 mM ADP and incubation on ice for 10 min. Methylation of the remodeled products was performed by incubation at 37°C for 15 min after addition of 5 U M.SssI and S-adenosylmethionine to a final concentration of 160 μM (New England BioLabs).

    Techniques: Electrophoresis, Incubation, Methylation, Nucleic Acid Electrophoresis, Plasmid Preparation, Staining, Amplification, Polymerase Chain Reaction, Clone Assay, Generated, Concentration Assay

    Duplex RNA promotes PRC2 activity. a , Native gel of di-nucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40-bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. b , Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4 and AEBP2, analyzed by SDS-PAGE and stained with Coomassie blue. c , Histone methyltransferase assay (HMTase assay) was performed with recombinant PRC2 complex, di-nucleosomes, S-Adenosylmethionine (SAM) with and without the co-factor JARID2 (amino acids 119-574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. d , Native 0.5X TBE gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration.

    Journal: bioRxiv

    Article Title: RNA matchmaking remodels lncRNA structure and promotes PRC2 activity

    doi: 10.1101/2020.04.13.040071

    Figure Lengend Snippet: Duplex RNA promotes PRC2 activity. a , Native gel of di-nucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40-bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. b , Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4 and AEBP2, analyzed by SDS-PAGE and stained with Coomassie blue. c , Histone methyltransferase assay (HMTase assay) was performed with recombinant PRC2 complex, di-nucleosomes, S-Adenosylmethionine (SAM) with and without the co-factor JARID2 (amino acids 119-574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. d , Native 0.5X TBE gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration.

    Article Snippet: Histone methyltransferase assays HMTase assays were performed in a total volume of 15 μl containing HMTase buffer (10 mM HEPES, pH 7.5, 2.5 mM MgCl2 , 0.25 mM EDTA, 4% Glycerol and 0.1 mM DTT) with 75 μM S-Adenosylmethionine (SAM, NEB), varying amounts ssRNA and duplex RNA (see above), 600 nM JARID2, 360 nM of dinucleosomes, and 600-660 nM recombinant human PRC2 complexes under the following conditions.

    Techniques: Activity Assay, Staining, Recombinant, SDS Page, HMT Assay, Western Blot, Titration