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  • 95
    Name:
    CpG Methyltransferase M SssI
    Description:
    CpG Methyltransferase M SssI 500 units
    Catalog Number:
    m0226l
    Price:
    296
    Size:
    500 units
    Category:
    DNA Methylases
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    Structured Review

    New England Biolabs cpg
    CpG Methyltransferase M SssI
    CpG Methyltransferase M SssI 500 units
    https://www.bioz.com/result/cpg/product/New England Biolabs
    Average 95 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    cpg - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Genome-wide methylation profiling of the different stages of hepatitis B virus-related hepatocellular carcinoma development in plasma cell-free DNA reveals potential biomarkers for early detection and high-risk monitoring of hepatocellular carcinoma"

    Article Title: Genome-wide methylation profiling of the different stages of hepatitis B virus-related hepatocellular carcinoma development in plasma cell-free DNA reveals potential biomarkers for early detection and high-risk monitoring of hepatocellular carcinoma

    Journal: Clinical Epigenetics

    doi: 10.1186/1868-7083-6-30

    Representative results of the quantitations of the methylation levels by Multiplex-BSP-seq for health control (HC), chronic hepatitis B infection(CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) plasma DNA samples. For each gene, the heat map of the methylation patterns for each CpG is shown. The methylation level (%) measured at each individual CpG site is expressed by the percentage of methylated CpG versus unmethylated CpG sites. HC, CHB, LC and HCC are represented by colored areas of blue, green, violet and red, respectively. The colored area is defined by 25%/75% quantiles. The comparison of the CpG methylation levels for each stage is colored blue to red, in small squares, to indicate the different P values. Representative heat map and methylation plot analysis of 5 target genes; consistently low levels of methylation of all CpGs were observed in GAPDH and steady high levels of CpG(CG1,2 and 7) methylation in KCNV1 are independent of the HCC developmental stage, while the methylation statuses of the other 3 genes (ZNF300, SLC22A20 and SHISA7) varied according to the developmental stage.
    Figure Legend Snippet: Representative results of the quantitations of the methylation levels by Multiplex-BSP-seq for health control (HC), chronic hepatitis B infection(CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) plasma DNA samples. For each gene, the heat map of the methylation patterns for each CpG is shown. The methylation level (%) measured at each individual CpG site is expressed by the percentage of methylated CpG versus unmethylated CpG sites. HC, CHB, LC and HCC are represented by colored areas of blue, green, violet and red, respectively. The colored area is defined by 25%/75% quantiles. The comparison of the CpG methylation levels for each stage is colored blue to red, in small squares, to indicate the different P values. Representative heat map and methylation plot analysis of 5 target genes; consistently low levels of methylation of all CpGs were observed in GAPDH and steady high levels of CpG(CG1,2 and 7) methylation in KCNV1 are independent of the HCC developmental stage, while the methylation statuses of the other 3 genes (ZNF300, SLC22A20 and SHISA7) varied according to the developmental stage.

    Techniques Used: Methylation, Multiplex Assay, Infection, CpG Methylation Assay

    Receiver operating characteristics (ROC) and multiple univariate logistic regression analyses for using CpGs to distinguish between hepatocellular carcinoma (HCC) developmental stages are shown according to the CpG position and disease stage (HC + CHB versus LC + HCC or HC + CHB + LC versus HCC). (A) Receiver operating characteristic (ROC) curves for ZNF300, SLC22A20 and SHISA7. Complete DNA methylation data from all four stages of HCC development were used to construct the ROC curves. The ROC curves plot the sensitivity versus 100-specificity. Upper panel: a lower cut-off value was used to distinguish between (LC + HCC)/ (HC + CHB).Lower panel: a higher cut-off value was used to distinguish between HCC/(HC + CHB + LC). (B) A multiple univariate logistic regression analysis was performed using the CpG methylation patterns to evaluate the association between gene methylation and the stage of HCC development. Relationship between the CpG methylation (odds ratios) and the developmental stage. To separate (LC + HCC)/ (HC + CHB) and HCC/(HC + CHB + LC), both univariate (which considers the methylation levels) and multivariate (which also considers the age and gender) logistic regressions were performed using CpG methylation data for ZNF300, SLC22A20 and SHISA7.
    Figure Legend Snippet: Receiver operating characteristics (ROC) and multiple univariate logistic regression analyses for using CpGs to distinguish between hepatocellular carcinoma (HCC) developmental stages are shown according to the CpG position and disease stage (HC + CHB versus LC + HCC or HC + CHB + LC versus HCC). (A) Receiver operating characteristic (ROC) curves for ZNF300, SLC22A20 and SHISA7. Complete DNA methylation data from all four stages of HCC development were used to construct the ROC curves. The ROC curves plot the sensitivity versus 100-specificity. Upper panel: a lower cut-off value was used to distinguish between (LC + HCC)/ (HC + CHB).Lower panel: a higher cut-off value was used to distinguish between HCC/(HC + CHB + LC). (B) A multiple univariate logistic regression analysis was performed using the CpG methylation patterns to evaluate the association between gene methylation and the stage of HCC development. Relationship between the CpG methylation (odds ratios) and the developmental stage. To separate (LC + HCC)/ (HC + CHB) and HCC/(HC + CHB + LC), both univariate (which considers the methylation levels) and multivariate (which also considers the age and gender) logistic regressions were performed using CpG methylation data for ZNF300, SLC22A20 and SHISA7.

    Techniques Used: DNA Methylation Assay, Construct, CpG Methylation Assay, Methylation

    Related Articles

    Clone Assay:

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *
    Article Snippet: Paragraph title: Cloning and in Vitro Methylation of pCpG Free-L1 Vector ... Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine.

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: The gene promoter fragments were amplified using the primers listed below and cloned into the pGL3 basic vector using the restriction enzymes XhoI and HindIII (NEB, Ipswich, United States). .. For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB).

    Amplification:

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *
    Article Snippet: For the construction of pCpG free L1 vector, the DNA fragment containing −1741/+56 region of the human NPC1L1 promoter was amplified using forward 5-ATCGATGCGAGTACTTGGACTCTATCTCTCTGTGG-3′ and reverse 5-ATCGATGCGAAGCTTCCCAGGTCTGGGAAGGGGTCA-3′ primers, and the amplified promoter fragments were then inserted into a promoterless CpG free vector (pCpG free basic lucia, InvivoGen, San Diego) upstream of the lucia reporter gene. .. Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: .. In vitro methylation assay An amplified Oct4 promoter product (500 ng) was methylated by M.SssI methylase (M0226S)(New England Biolabs, UK), at 37°C for 3 h, with the subsequent inactivation of enzyme at 60°C for 20 min. To identify of the methylation state of Oct4 promoter, the methylated product was digested with Msp I (R057S)(Enzynomics, Korea) and Hpa II (R049S)(Enzynomics, Korea). .. Screening for the methylated Oct4 promoter binding protein A vector containing mouse Oct4 promoter was constructed.

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: The gene promoter fragments were amplified using the primers listed below and cloned into the pGL3 basic vector using the restriction enzymes XhoI and HindIII (NEB, Ipswich, United States). .. For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB).

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: .. An amplified Oct4 promoter product (500 ng) was methylated by M.SssI methylase (M0226S)(New England Biolabs, UK), at 37°C for 3 h, with the subsequent inactivation of enzyme at 60°C for 20 min. To identify of the methylation state of Oct4 promoter, the methylated product was digested with Msp I (R057S)(Enzynomics, Korea) and Hpa II (R049S)(Enzynomics, Korea). .. A vector containing mouse Oct4 promoter was constructed.

    Reporter Assay:

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: Paragraph title: Luciferase reporter assay and cell lines ... For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB).

    Positive Control:

    Article Title: Aberrant methylation of DACT1 and DACT2 are associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma
    Article Snippet: .. Genomic DNA, which was treated by CpG methyltransferase (Sss I) following the manufacturer’s directions (New England BioLabs, Inc, Beverly, MA), was used as positive control. ..

    Article Title: Differential Expression of ADAM23, CDKN2A (P16), MMP14 and VIM Associated with Giant Cell Tumor of Bone
    Article Snippet: .. Bisulfite-modified DNA from peripheral blood lymphocytes from a healthy individual was previously treated and untreated with CpG methyltransferase (M.SssI) (New England Biolabs, Ipswich, MA, USA) and served as a positive control for hypermethylated and unmethylated DNA. .. A blank control containing all the PCR components (except template DNA) was also included in all of the experiments.

    Construct:

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *
    Article Snippet: .. Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine. .. Plasmid DNA was then purified by using Qiagen miniprep kit and quantified using a spectrophotometer.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: A vector containing mouse Oct4 promoter was constructed. .. The purified DNA was methylated artificially with CpG Methyltransferase (M.SssI;(M0226S)(New England Biolabs, UK)), and biotinylated with biotin-labeled dCTP and Klenow enzyme.

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: .. For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB). .. For the Luciferase Assay the cells were seeded into 48-well plates.

    Article Title: Induction of DNA Demethylation Depending on Two Sets of Sox2 and Adjacent Oct3/4 Binding Sites (Sox-Oct Motifs) within the Mouse H19/Insulin-like Growth Factor 2 (Igf2
    Article Snippet: .. Constructs used in the experiments were methylated in vitro with bacterial HpaII methyltransferase (M.HpaII; New England Biolabs), HhaI methyltransferase (M.HhaI; New England Biolabs), and CpG methyltransferase (M.SssI; New England Biolabs). .. Prior to transfection, methylation was confirmed by digestion with 8–10 units of HpaII or HhaI restriction endonucleases per 1 μg of methylated plasmid.

    Electrophoresis:

    Article Title: Differential Expression of ADAM23, CDKN2A (P16), MMP14 and VIM Associated with Giant Cell Tumor of Bone
    Article Snippet: Bisulfite-modified DNA from peripheral blood lymphocytes from a healthy individual was previously treated and untreated with CpG methyltransferase (M.SssI) (New England Biolabs, Ipswich, MA, USA) and served as a positive control for hypermethylated and unmethylated DNA. .. Reaction products were separated using electrophoresis on an 8% polyacrylamide gel and stained with silver nitrate.

    Microarray:

    Article Title: Schizophrenia-Like Phenotype Inherited by the F2 Generation of a Gestational Disruption Model of Schizophrenia
    Article Snippet: Microarray analysis of DNA methylation was performed using methylated DNA immunoprecipitation (MeDIP)-chip by a commercial vendor (ArrayStar, Rockville, Maryland). .. Given that a fully methylated profile is required to determine an absolute methylation score, DNA from each sample was pooled and treated with CpG methyltransferase (M.SssI, NEB).

    Incubation:

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma
    Article Snippet: .. The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned. .. The DNA binding reaction was carried out in a 20 μl reaction mixture containing Gel Shift Binding Buffer (Promega), 10 μg nuclear extract and the biotin-labelled probe or methylated biotin-labelled probe, with or without 100-fold molar excess unlabelled competitor.

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *
    Article Snippet: .. Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine. .. Plasmid DNA was then purified by using Qiagen miniprep kit and quantified using a spectrophotometer.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: The purified DNA was methylated artificially with CpG Methyltransferase (M.SssI;(M0226S)(New England Biolabs, UK)), and biotinylated with biotin-labeled dCTP and Klenow enzyme. .. The DNA was then incubated with the mESC protein extract.

    Article Title: RNautophagy/DNautophagy possesses selectivity for RNA/DNA substrates
    Article Snippet: .. Briefly, 4 nmol of dsDNA was incubated with S-adenosylmethionine (NEB) and CpG methyltransferase in NE Buffer (NEB) at 37°C. .. After 8 h of incubation, methylated DNA was purified using phenol-chloroform, followed by ethanol precipitation.

    Luciferase:

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: Paragraph title: Luciferase reporter assay and cell lines ... For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB).

    Modification:

    Article Title: Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation
    Article Snippet: In vitro methylation of plasmid DNA Methylated plasmids were generated by incubating 1 μg of plasmid DNA with 4 units/μl of the CpG methyltransferase, M.Sss I (New England BioLabs), according to the manufacturer's instructions. .. Complete methylation was verified by plasmid DNA bisulfite modification and pyrosequencing using specific primers.

    Article Title: Differential Expression of ADAM23, CDKN2A (P16), MMP14 and VIM Associated with Giant Cell Tumor of Bone
    Article Snippet: Paragraph title: Bisulfite Modification of DNA and Methylation-Specific PCR ... Bisulfite-modified DNA from peripheral blood lymphocytes from a healthy individual was previously treated and untreated with CpG methyltransferase (M.SssI) (New England Biolabs, Ipswich, MA, USA) and served as a positive control for hypermethylated and unmethylated DNA.

    Transformation Assay:

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: The human epithelial, embryonal kidney cell line HEK 293 T, established from a human primary embryonal kidney transformed by adenovirus type 5 was cultured according to the manufacturer’s recommendation in high glucose DMEM GLUTAMAX (Gibco, Waltham, United States) containing 10% FCS (Gibco) and 1% penicillin and streptomycin at 37 °C and with 5% CO2. .. For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB).

    Transfection:

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB). .. Lipofectamine 2000 (Invitrogen) was used as transfection reagent according to the manufacturer’s suggestion.

    Article Title: Methylation Regulates Hepatitis B Viral Protein Expression
    Article Snippet: .. The CpG methyltransferase used for in vitro methylation of the transfected HBV DNA targets all CpG dinucleotides. ..

    Article Title: Induction of DNA Demethylation Depending on Two Sets of Sox2 and Adjacent Oct3/4 Binding Sites (Sox-Oct Motifs) within the Mouse H19/Insulin-like Growth Factor 2 (Igf2
    Article Snippet: Constructs used in the experiments were methylated in vitro with bacterial HpaII methyltransferase (M.HpaII; New England Biolabs), HhaI methyltransferase (M.HhaI; New England Biolabs), and CpG methyltransferase (M.SssI; New England Biolabs). .. Prior to transfection, methylation was confirmed by digestion with 8–10 units of HpaII or HhaI restriction endonucleases per 1 μg of methylated plasmid.

    Genomic Sequencing:

    Article Title: Aberrant methylation of DACT1 and DACT2 are associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma
    Article Snippet: Paragraph title: Methylation analysis of DACT via methylation-specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS) methods ... Genomic DNA, which was treated by CpG methyltransferase (Sss I) following the manufacturer’s directions (New England BioLabs, Inc, Beverly, MA), was used as positive control.

    Cell Culture:

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: The human epithelial, embryonal kidney cell line HEK 293 T, established from a human primary embryonal kidney transformed by adenovirus type 5 was cultured according to the manufacturer’s recommendation in high glucose DMEM GLUTAMAX (Gibco, Waltham, United States) containing 10% FCS (Gibco) and 1% penicillin and streptomycin at 37 °C and with 5% CO2. .. For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB).

    Generated:

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma
    Article Snippet: The mutated probe of the CD147 promoter was generated by the same procedure, except that several oligonucleotides were mutated (The sequences were listed in ) in the core sequence of the Sp1-binding site. .. The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned.

    Article Title: Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation
    Article Snippet: .. In vitro methylation of plasmid DNA Methylated plasmids were generated by incubating 1 μg of plasmid DNA with 4 units/μl of the CpG methyltransferase, M.Sss I (New England BioLabs), according to the manufacturer's instructions. .. Complete methylation was verified by plasmid DNA bisulfite modification and pyrosequencing using specific primers.

    Polymerase Chain Reaction:

    Article Title: Aberrant methylation of DACT1 and DACT2 are associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma
    Article Snippet: Paragraph title: Methylation analysis of DACT via methylation-specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS) methods ... Genomic DNA, which was treated by CpG methyltransferase (Sss I) following the manufacturer’s directions (New England BioLabs, Inc, Beverly, MA), was used as positive control.

    Article Title: Differential Expression of ADAM23, CDKN2A (P16), MMP14 and VIM Associated with Giant Cell Tumor of Bone
    Article Snippet: Paragraph title: Bisulfite Modification of DNA and Methylation-Specific PCR ... Bisulfite-modified DNA from peripheral blood lymphocytes from a healthy individual was previously treated and untreated with CpG methyltransferase (M.SssI) (New England Biolabs, Ipswich, MA, USA) and served as a positive control for hypermethylated and unmethylated DNA.

    Sonication:

    Article Title: Schizophrenia-Like Phenotype Inherited by the F2 Generation of a Gestational Disruption Model of Schizophrenia
    Article Snippet: In brief, genomic DNA was extracted and sonicated to random fragments of 200–1000 bp. .. Given that a fully methylated profile is required to determine an absolute methylation score, DNA from each sample was pooled and treated with CpG methyltransferase (M.SssI, NEB).

    Recombinant:

    Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos
    Article Snippet: Paragraph title: Methylation of the Recombinant Plasmids ... The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), CpG methyltransferase (M. SssI, 4 U/ul, 2 ul) (New England Biolabs) and H2 O (33 ul).

    Methylated DNA Immunoprecipitation:

    Article Title: Schizophrenia-Like Phenotype Inherited by the F2 Generation of a Gestational Disruption Model of Schizophrenia
    Article Snippet: Microarray analysis of DNA methylation was performed using methylated DNA immunoprecipitation (MeDIP)-chip by a commercial vendor (ArrayStar, Rockville, Maryland). .. Given that a fully methylated profile is required to determine an absolute methylation score, DNA from each sample was pooled and treated with CpG methyltransferase (M.SssI, NEB).

    Methylation:

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma
    Article Snippet: .. The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned. .. The DNA binding reaction was carried out in a 20 μl reaction mixture containing Gel Shift Binding Buffer (Promega), 10 μg nuclear extract and the biotin-labelled probe or methylated biotin-labelled probe, with or without 100-fold molar excess unlabelled competitor.

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *
    Article Snippet: Paragraph title: Cloning and in Vitro Methylation of pCpG Free-L1 Vector ... Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: .. The purified DNA was methylated artificially with CpG Methyltransferase (M.SssI;(M0226S)(New England Biolabs, UK)), and biotinylated with biotin-labeled dCTP and Klenow enzyme. .. Biotinylated DNA was attached to streptavidin with Dynabeads kilobaseBINDER kit (60101)(Invitrogen, USA).

    Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos
    Article Snippet: .. The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), CpG methyltransferase (M. SssI, 4 U/ul, 2 ul) (New England Biolabs) and H2 O (33 ul). .. The DNA was purified by phenol-chloroform extraction and ethanol precipitation.

    Article Title: Aberrant methylation of DACT1 and DACT2 are associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma
    Article Snippet: Paragraph title: Methylation analysis of DACT via methylation-specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS) methods ... Genomic DNA, which was treated by CpG methyltransferase (Sss I) following the manufacturer’s directions (New England BioLabs, Inc, Beverly, MA), was used as positive control.

    Article Title: RNautophagy/DNautophagy possesses selectivity for RNA/DNA substrates
    Article Snippet: Paragraph title: Methylation of DNA ... Briefly, 4 nmol of dsDNA was incubated with S-adenosylmethionine (NEB) and CpG methyltransferase in NE Buffer (NEB) at 37°C.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: .. In vitro methylation assay An amplified Oct4 promoter product (500 ng) was methylated by M.SssI methylase (M0226S)(New England Biolabs, UK), at 37°C for 3 h, with the subsequent inactivation of enzyme at 60°C for 20 min. To identify of the methylation state of Oct4 promoter, the methylated product was digested with Msp I (R057S)(Enzynomics, Korea) and Hpa II (R049S)(Enzynomics, Korea). .. Screening for the methylated Oct4 promoter binding protein A vector containing mouse Oct4 promoter was constructed.

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: .. For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB). .. For the Luciferase Assay the cells were seeded into 48-well plates.

    Article Title: Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation
    Article Snippet: .. In vitro methylation of plasmid DNA Methylated plasmids were generated by incubating 1 μg of plasmid DNA with 4 units/μl of the CpG methyltransferase, M.Sss I (New England BioLabs), according to the manufacturer's instructions. .. Complete methylation was verified by plasmid DNA bisulfite modification and pyrosequencing using specific primers.

    Article Title: Differential Expression of ADAM23, CDKN2A (P16), MMP14 and VIM Associated with Giant Cell Tumor of Bone
    Article Snippet: Paragraph title: Bisulfite Modification of DNA and Methylation-Specific PCR ... Bisulfite-modified DNA from peripheral blood lymphocytes from a healthy individual was previously treated and untreated with CpG methyltransferase (M.SssI) (New England Biolabs, Ipswich, MA, USA) and served as a positive control for hypermethylated and unmethylated DNA.

    Article Title: Methylation Regulates Hepatitis B Viral Protein Expression
    Article Snippet: .. The CpG methyltransferase used for in vitro methylation of the transfected HBV DNA targets all CpG dinucleotides. ..

    Article Title: Induction of DNA Demethylation Depending on Two Sets of Sox2 and Adjacent Oct3/4 Binding Sites (Sox-Oct Motifs) within the Mouse H19/Insulin-like Growth Factor 2 (Igf2
    Article Snippet: .. Constructs used in the experiments were methylated in vitro with bacterial HpaII methyltransferase (M.HpaII; New England Biolabs), HhaI methyltransferase (M.HhaI; New England Biolabs), and CpG methyltransferase (M.SssI; New England Biolabs). .. Prior to transfection, methylation was confirmed by digestion with 8–10 units of HpaII or HhaI restriction endonucleases per 1 μg of methylated plasmid.

    Article Title: Schizophrenia-Like Phenotype Inherited by the F2 Generation of a Gestational Disruption Model of Schizophrenia
    Article Snippet: .. Given that a fully methylated profile is required to determine an absolute methylation score, DNA from each sample was pooled and treated with CpG methyltransferase (M.SssI, NEB). .. The immunoprecipitated DNA was eluted and purified by phenol–chloroform extraction and ethanol precipitation.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: .. An amplified Oct4 promoter product (500 ng) was methylated by M.SssI methylase (M0226S)(New England Biolabs, UK), at 37°C for 3 h, with the subsequent inactivation of enzyme at 60°C for 20 min. To identify of the methylation state of Oct4 promoter, the methylated product was digested with Msp I (R057S)(Enzynomics, Korea) and Hpa II (R049S)(Enzynomics, Korea). .. A vector containing mouse Oct4 promoter was constructed.

    Electrophoretic Mobility Shift Assay:

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma
    Article Snippet: Paragraph title: Electrophoretic mobility shift assays (EMSA) ... The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned.

    Purification:

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *
    Article Snippet: Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine. .. Plasmid DNA was then purified by using Qiagen miniprep kit and quantified using a spectrophotometer.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: .. The purified DNA was methylated artificially with CpG Methyltransferase (M.SssI;(M0226S)(New England Biolabs, UK)), and biotinylated with biotin-labeled dCTP and Klenow enzyme. .. Biotinylated DNA was attached to streptavidin with Dynabeads kilobaseBINDER kit (60101)(Invitrogen, USA).

    Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos
    Article Snippet: The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), CpG methyltransferase (M. SssI, 4 U/ul, 2 ul) (New England Biolabs) and H2 O (33 ul). .. The DNA was purified by phenol-chloroform extraction and ethanol precipitation.

    Article Title: RNautophagy/DNautophagy possesses selectivity for RNA/DNA substrates
    Article Snippet: Briefly, 4 nmol of dsDNA was incubated with S-adenosylmethionine (NEB) and CpG methyltransferase in NE Buffer (NEB) at 37°C. .. After 8 h of incubation, methylated DNA was purified using phenol-chloroform, followed by ethanol precipitation.

    Article Title: Schizophrenia-Like Phenotype Inherited by the F2 Generation of a Gestational Disruption Model of Schizophrenia
    Article Snippet: Given that a fully methylated profile is required to determine an absolute methylation score, DNA from each sample was pooled and treated with CpG methyltransferase (M.SssI, NEB). .. The immunoprecipitated DNA was eluted and purified by phenol–chloroform extraction and ethanol precipitation.

    Sequencing:

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma
    Article Snippet: The mutated probe of the CD147 promoter was generated by the same procedure, except that several oligonucleotides were mutated (The sequences were listed in ) in the core sequence of the Sp1-binding site. .. The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned.

    Article Title: Aberrant methylation of DACT1 and DACT2 are associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma
    Article Snippet: Based on this potential difference in the DNA sequence between methylated and unmethylated alleles after bisulfite treatment, we designed MSP primers to analyze two regions of DACT1 , and one region of DACT2 and DACT3 . .. Genomic DNA, which was treated by CpG methyltransferase (Sss I) following the manufacturer’s directions (New England BioLabs, Inc, Beverly, MA), was used as positive control.

    Labeling:

    Article Title: Schizophrenia-Like Phenotype Inherited by the F2 Generation of a Gestational Disruption Model of Schizophrenia
    Article Snippet: Given that a fully methylated profile is required to determine an absolute methylation score, DNA from each sample was pooled and treated with CpG methyltransferase (M.SssI, NEB). .. The total input and immunoprecipitated DNA were labeled with Cy3- and Cy5-labeled random 9-mers, respectively, and were hybridized to NimbleGen Rat DNA Methylation 385 K Promoter Plus CpG Island Array, which is a 385-k format array design containing 15 809 CpG Islands and all well-characterized promoter regions (from about −1300 to +500 bp of the TSSs).

    Demethylation Assay:

    Article Title: Induction of DNA Demethylation Depending on Two Sets of Sox2 and Adjacent Oct3/4 Binding Sites (Sox-Oct Motifs) within the Mouse H19/Insulin-like Growth Factor 2 (Igf2
    Article Snippet: Paragraph title: DNA Demethylation Assay ... Constructs used in the experiments were methylated in vitro with bacterial HpaII methyltransferase (M.HpaII; New England Biolabs), HhaI methyltransferase (M.HhaI; New England Biolabs), and CpG methyltransferase (M.SssI; New England Biolabs).

    Plasmid Preparation:

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *
    Article Snippet: .. Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine. .. Plasmid DNA was then purified by using Qiagen miniprep kit and quantified using a spectrophotometer.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: The DNA vector was treated with the appropriate restriction enzyme, and only the mouse Oct4 promoter was purified. .. The purified DNA was methylated artificially with CpG Methyltransferase (M.SssI;(M0226S)(New England Biolabs, UK)), and biotinylated with biotin-labeled dCTP and Klenow enzyme.

    Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos
    Article Snippet: .. The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), CpG methyltransferase (M. SssI, 4 U/ul, 2 ul) (New England Biolabs) and H2 O (33 ul). .. The DNA was purified by phenol-chloroform extraction and ethanol precipitation.

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: The gene promoter fragments were amplified using the primers listed below and cloned into the pGL3 basic vector using the restriction enzymes XhoI and HindIII (NEB, Ipswich, United States). .. For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB).

    Article Title: Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation
    Article Snippet: .. In vitro methylation of plasmid DNA Methylated plasmids were generated by incubating 1 μg of plasmid DNA with 4 units/μl of the CpG methyltransferase, M.Sss I (New England BioLabs), according to the manufacturer's instructions. .. Complete methylation was verified by plasmid DNA bisulfite modification and pyrosequencing using specific primers.

    Article Title: Induction of DNA Demethylation Depending on Two Sets of Sox2 and Adjacent Oct3/4 Binding Sites (Sox-Oct Motifs) within the Mouse H19/Insulin-like Growth Factor 2 (Igf2
    Article Snippet: Constructs used in the experiments were methylated in vitro with bacterial HpaII methyltransferase (M.HpaII; New England Biolabs), HhaI methyltransferase (M.HhaI; New England Biolabs), and CpG methyltransferase (M.SssI; New England Biolabs). .. Prior to transfection, methylation was confirmed by digestion with 8–10 units of HpaII or HhaI restriction endonucleases per 1 μg of methylated plasmid.

    Binding Assay:

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma
    Article Snippet: The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned. .. The DNA binding reaction was carried out in a 20 μl reaction mixture containing Gel Shift Binding Buffer (Promega), 10 μg nuclear extract and the biotin-labelled probe or methylated biotin-labelled probe, with or without 100-fold molar excess unlabelled competitor.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: Paragraph title: Screening for the methylated Oct4 promoter binding protein ... The purified DNA was methylated artificially with CpG Methyltransferase (M.SssI;(M0226S)(New England Biolabs, UK)), and biotinylated with biotin-labeled dCTP and Klenow enzyme.

    In Vitro:

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *
    Article Snippet: Paragraph title: Cloning and in Vitro Methylation of pCpG Free-L1 Vector ... Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: .. In vitro methylation assay An amplified Oct4 promoter product (500 ng) was methylated by M.SssI methylase (M0226S)(New England Biolabs, UK), at 37°C for 3 h, with the subsequent inactivation of enzyme at 60°C for 20 min. To identify of the methylation state of Oct4 promoter, the methylated product was digested with Msp I (R057S)(Enzynomics, Korea) and Hpa II (R049S)(Enzynomics, Korea). .. Screening for the methylated Oct4 promoter binding protein A vector containing mouse Oct4 promoter was constructed.

    Article Title: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis
    Article Snippet: .. For analysis of the influence of promoter methylation the constructs were in vitro methylated by using the CpG methyltransferase M. SssI (NEB). .. For the Luciferase Assay the cells were seeded into 48-well plates.

    Article Title: Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation
    Article Snippet: .. In vitro methylation of plasmid DNA Methylated plasmids were generated by incubating 1 μg of plasmid DNA with 4 units/μl of the CpG methyltransferase, M.Sss I (New England BioLabs), according to the manufacturer's instructions. .. Complete methylation was verified by plasmid DNA bisulfite modification and pyrosequencing using specific primers.

    Article Title: Methylation Regulates Hepatitis B Viral Protein Expression
    Article Snippet: .. The CpG methyltransferase used for in vitro methylation of the transfected HBV DNA targets all CpG dinucleotides. ..

    Article Title: Induction of DNA Demethylation Depending on Two Sets of Sox2 and Adjacent Oct3/4 Binding Sites (Sox-Oct Motifs) within the Mouse H19/Insulin-like Growth Factor 2 (Igf2
    Article Snippet: .. Constructs used in the experiments were methylated in vitro with bacterial HpaII methyltransferase (M.HpaII; New England Biolabs), HhaI methyltransferase (M.HhaI; New England Biolabs), and CpG methyltransferase (M.SssI; New England Biolabs). .. Prior to transfection, methylation was confirmed by digestion with 8–10 units of HpaII or HhaI restriction endonucleases per 1 μg of methylated plasmid.

    Article Title: Imprinted gene Zinc finger protein 127 is a novel regulator of master pluripotency transcription factor, Oct4
    Article Snippet: Paragraph title: In vitro methylation assay ... An amplified Oct4 promoter product (500 ng) was methylated by M.SssI methylase (M0226S)(New England Biolabs, UK), at 37°C for 3 h, with the subsequent inactivation of enzyme at 60°C for 20 min. To identify of the methylation state of Oct4 promoter, the methylated product was digested with Msp I (R057S)(Enzynomics, Korea) and Hpa II (R049S)(Enzynomics, Korea).

    Ethanol Precipitation:

    Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos
    Article Snippet: The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), CpG methyltransferase (M. SssI, 4 U/ul, 2 ul) (New England Biolabs) and H2 O (33 ul). .. The DNA was purified by phenol-chloroform extraction and ethanol precipitation.

    Article Title: RNautophagy/DNautophagy possesses selectivity for RNA/DNA substrates
    Article Snippet: Briefly, 4 nmol of dsDNA was incubated with S-adenosylmethionine (NEB) and CpG methyltransferase in NE Buffer (NEB) at 37°C. .. After 8 h of incubation, methylated DNA was purified using phenol-chloroform, followed by ethanol precipitation.

    Article Title: Schizophrenia-Like Phenotype Inherited by the F2 Generation of a Gestational Disruption Model of Schizophrenia
    Article Snippet: Given that a fully methylated profile is required to determine an absolute methylation score, DNA from each sample was pooled and treated with CpG methyltransferase (M.SssI, NEB). .. The immunoprecipitated DNA was eluted and purified by phenol–chloroform extraction and ethanol precipitation.

    Spectrophotometry:

    Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *
    Article Snippet: Briefly, 1 μg of plasmid DNA was added to a reaction containing CpG methyltransferase (Sss1, 4 units/μl) in the presence of 160 μ m S -adenosylmethionine (New England Biolabs) and incubated for 4 h at 37 °C, and S -adenosylmethionine was replenished after every 2 h. Unmethylated control reaction contained the NPC1L1 promoter construct and methylases but not S -adenosylmethionine. .. Plasmid DNA was then purified by using Qiagen miniprep kit and quantified using a spectrophotometer.

    DNA Methylation Assay:

    Article Title: Differential Expression of ADAM23, CDKN2A (P16), MMP14 and VIM Associated with Giant Cell Tumor of Bone
    Article Snippet: The DNA methylation status in the CpG island promoter was determined using the previously described MSP procedure, which used primers specific for the methylated (M) or unmethylated (U) sequences of the bisulfite-modified DNA. .. Bisulfite-modified DNA from peripheral blood lymphocytes from a healthy individual was previously treated and untreated with CpG methyltransferase (M.SssI) (New England Biolabs, Ipswich, MA, USA) and served as a positive control for hypermethylated and unmethylated DNA.

    Article Title: Schizophrenia-Like Phenotype Inherited by the F2 Generation of a Gestational Disruption Model of Schizophrenia
    Article Snippet: Microarray analysis of DNA methylation was performed using methylated DNA immunoprecipitation (MeDIP)-chip by a commercial vendor (ArrayStar, Rockville, Maryland). .. Given that a fully methylated profile is required to determine an absolute methylation score, DNA from each sample was pooled and treated with CpG methyltransferase (M.SssI, NEB).

    Immunoprecipitation:

    Article Title: Schizophrenia-Like Phenotype Inherited by the F2 Generation of a Gestational Disruption Model of Schizophrenia
    Article Snippet: Immunoprecipitation of methylated DNA was performed using Biomag magnetic bead-coupled mouse monoclonal antibody against 5-methylcytidine. .. Given that a fully methylated profile is required to determine an absolute methylation score, DNA from each sample was pooled and treated with CpG methyltransferase (M.SssI, NEB).

    Staining:

    Article Title: Differential Expression of ADAM23, CDKN2A (P16), MMP14 and VIM Associated with Giant Cell Tumor of Bone
    Article Snippet: Bisulfite-modified DNA from peripheral blood lymphocytes from a healthy individual was previously treated and untreated with CpG methyltransferase (M.SssI) (New England Biolabs, Ipswich, MA, USA) and served as a positive control for hypermethylated and unmethylated DNA. .. Reaction products were separated using electrophoresis on an 8% polyacrylamide gel and stained with silver nitrate.

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    New England Biolabs cpg methyltransferase m sssi
    Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated <t>CpG</t> reporter constructs. CD147P/pGL3 treated or untreated with <t>SssI</t> methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P
    Cpg Methyltransferase M Sssi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma

    doi: 10.1111/j.1582-4934.2010.01124.x

    Figure Lengend Snippet: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P

    Article Snippet: The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned.

    Techniques: Methylation, Binding Assay, Activity Assay, In Vitro, Electrophoretic Mobility Shift Assay, Mutagenesis, DNA Methylation Assay, Transfection, Construct, Luciferase

    DNA methylation of Gpx1 decreases gene expression. In vitro methylation of Gpx1 target region inhibited transcriptional activity, as measured by a luciferase reporter assay. (A) Schematic representation of the plasmid construction containing the Gpx1 CpG island region. (B) Luciferase activity ratio of methylated (M.SssI treated) to unmethylated control (CTL) plasmids containing a CpG-free promoter or the Gpx1 CpG island region. The assay was repeated 4 times and data are mean ± SEM. *: p

    Journal: PLoS ONE

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice

    doi: 10.1371/journal.pone.0151526

    Figure Lengend Snippet: DNA methylation of Gpx1 decreases gene expression. In vitro methylation of Gpx1 target region inhibited transcriptional activity, as measured by a luciferase reporter assay. (A) Schematic representation of the plasmid construction containing the Gpx1 CpG island region. (B) Luciferase activity ratio of methylated (M.SssI treated) to unmethylated control (CTL) plasmids containing a CpG-free promoter or the Gpx1 CpG island region. The assay was repeated 4 times and data are mean ± SEM. *: p

    Article Snippet: M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Techniques: DNA Methylation Assay, Expressing, In Vitro, Methylation, Activity Assay, Luciferase, Reporter Assay, Plasmid Preparation, CTL Assay

    Hypermethylated regions in LNCaP compared to PrEC cells can serve as biomarkers for prostate cancer . A , DNA methylation at representative regions identified as hypermethylated in LNCaP cells compared to PrEC cells was measured in prostate cell lines using the COMPARE-MS assay as described previously [ 9 ]. The extent of methylation at each region is color scaled from white to red as shown, with white representing absence of detectable methylation, and red representing nearly complete methylation of all input copies. M.SssI-treated, completely-methylated, WBC DNA served as a positive control. All of the prostate cancer cell lines showed a high degree of methylation at multiple regions, while the PrEC normal prostate cells did not show any detectable methylation at these regions as expected. B , COMPARE-MS analysis of DNA methylation at three of the five regions from (A) showed significant methylation of at least one of the three regions in every tumor sample with very low or undetectable methylation in the matched normal tissue samples.

    Journal: BMC Genomics

    Article Title: Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

    doi: 10.1186/1471-2164-12-313

    Figure Lengend Snippet: Hypermethylated regions in LNCaP compared to PrEC cells can serve as biomarkers for prostate cancer . A , DNA methylation at representative regions identified as hypermethylated in LNCaP cells compared to PrEC cells was measured in prostate cell lines using the COMPARE-MS assay as described previously [ 9 ]. The extent of methylation at each region is color scaled from white to red as shown, with white representing absence of detectable methylation, and red representing nearly complete methylation of all input copies. M.SssI-treated, completely-methylated, WBC DNA served as a positive control. All of the prostate cancer cell lines showed a high degree of methylation at multiple regions, while the PrEC normal prostate cells did not show any detectable methylation at these regions as expected. B , COMPARE-MS analysis of DNA methylation at three of the five regions from (A) showed significant methylation of at least one of the three regions in every tumor sample with very low or undetectable methylation in the matched normal tissue samples.

    Article Snippet: Treatment of DNA samples with M.HhaI and/or M.HpaII, and M.SssI DNA methyltransferases (NEB) were carried out according to the manufacturer's recommended protocol.

    Techniques: DNA Methylation Assay, Mass Spectrometry, Methylation, Positive Control

    Overview and pre-microarray performance of MBD-chip . A , Overview of MBD-chip. Genomic DNA is: i) fragmented (in this case with restriction enzymes), ii) enriched for methylated DNA using MBD2-MBD-magnetic beads, and iii) amplified, fragmented, labeled and hybridized to tiling microarrays. Comparison with a total input fraction allows identification of methylated regions. B , Degree of MBD2-MBD enrichment is non-linearly proportional to the number of methylated CpGs. WBC DNA was methylated at 0, 6, 10 or 37 CpG sites within an R.AluI restriction fragment within the GSTP1 promoter by treatment with M.HpaII +/- M.HhaI or with M.SssI or no enzymes. The degree of MBD2-MBD enrichment compared to mock (no MBD control), as measured by qPCR, was related to the number of methylated CpGs. ND, not detectable. C , The MBD-chip process enriches DNA with high density of methylated-CpGs. DNA from LNCaP and PrEC cells was completely methylated with M.SssI or left untreated. MBD2-MBD enrichment at regions in HBB and GSTP1 promoters, as examined by qPCR, are shown. The CpG density (Low, indicates

    Journal: BMC Genomics

    Article Title: Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

    doi: 10.1186/1471-2164-12-313

    Figure Lengend Snippet: Overview and pre-microarray performance of MBD-chip . A , Overview of MBD-chip. Genomic DNA is: i) fragmented (in this case with restriction enzymes), ii) enriched for methylated DNA using MBD2-MBD-magnetic beads, and iii) amplified, fragmented, labeled and hybridized to tiling microarrays. Comparison with a total input fraction allows identification of methylated regions. B , Degree of MBD2-MBD enrichment is non-linearly proportional to the number of methylated CpGs. WBC DNA was methylated at 0, 6, 10 or 37 CpG sites within an R.AluI restriction fragment within the GSTP1 promoter by treatment with M.HpaII +/- M.HhaI or with M.SssI or no enzymes. The degree of MBD2-MBD enrichment compared to mock (no MBD control), as measured by qPCR, was related to the number of methylated CpGs. ND, not detectable. C , The MBD-chip process enriches DNA with high density of methylated-CpGs. DNA from LNCaP and PrEC cells was completely methylated with M.SssI or left untreated. MBD2-MBD enrichment at regions in HBB and GSTP1 promoters, as examined by qPCR, are shown. The CpG density (Low, indicates

    Article Snippet: Treatment of DNA samples with M.HhaI and/or M.HpaII, and M.SssI DNA methyltransferases (NEB) were carried out according to the manufacturer's recommended protocol.

    Techniques: Microarray, Chromatin Immunoprecipitation, Methylation, Magnetic Beads, Amplification, Labeling, Real-time Polymerase Chain Reaction

    Reporter gene activities of human ERBB2 promoter constructs (A) Luciferase activities of progressive ERBB2 deletion constructs in A549 cells (see text for details). Normalized luciferase activities were expressed relative to the values from the −499 ERBB2 -pCpGL construct, which was assigned an arbitrary value of 100. (B) Effect of DNA methylation status on the luciferase activity of the −499 ERBB2 -pCpGL construct. CpG sites methylated by HpaII or SssI methyltransferase are indicated. Data represent the mean ± standard deviation of three independent experiments. *** p

    Journal: Gene

    Article Title: Investigation of the Role of DNA Methylation in the Expression of ERBB2 in Human Myocardium

    doi: 10.1016/j.gene.2017.07.058

    Figure Lengend Snippet: Reporter gene activities of human ERBB2 promoter constructs (A) Luciferase activities of progressive ERBB2 deletion constructs in A549 cells (see text for details). Normalized luciferase activities were expressed relative to the values from the −499 ERBB2 -pCpGL construct, which was assigned an arbitrary value of 100. (B) Effect of DNA methylation status on the luciferase activity of the −499 ERBB2 -pCpGL construct. CpG sites methylated by HpaII or SssI methyltransferase are indicated. Data represent the mean ± standard deviation of three independent experiments. *** p

    Article Snippet: The −499 ERBB2 -pCpGL reporter plasmid was methylated using the CpG methyltransferases M.SssI or HpaII (New England Biolabs, Massachusetts, USA).

    Techniques: Construct, Luciferase, DNA Methylation Assay, Activity Assay, Methylation, Standard Deviation