Home > Adenylate cyclase & > N6-methyladenosine

N6-methyladenosine

N6-甲基腺嘌呤,NSC-29409,m6A

N6-methyladenosine (m6A)是碱基修饰的腺苷类似物,在天然RNAs中作为稀有核苷。N6-methyladenosine(m6A)是mRNA的第五种碱基。

目录号
EY0317
EY0317
EY0317
纯度
99.11%
99.11%
99.11%
规格
50 mg
100 mg
500 mg
原价
590.00_
780.00_
1460
售价
590.00_
780.00_
1460
库存
现货
现货
现货
订购
订购
订购
订购
订购
订购
  • 生物活性

    N6-Methyladenosine is an abundant nucleoside in cellular mRNA that undergoes demethylation under physiological conditions by fat mass.

  • 体外研究

  • 体内研究

  • 激酶实验

  • 细胞实验

    Mammalian cell culture, siRNAknockdown and cell fractionation[1]

    Human cervical adenocarcinoma cell line HeLa (CCL-2) and human embryonic kidney (HEK) cell line HEK293T (CRL-11268) were cultured under standard conditions. Control siRNA, METTL3 siRNA, METTL14 siRNA or HNRNPG siRNA was transfected into HEK293T cells at a concentration of 20–50 nM, using Lipofectamine RNAiMAX. Cells were collected 48 h after transfection, shock-frozen in liquid nitrogen and stored at−80◦C for further studies. Nuclear and cytoplasmic extracts were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents

    Western blotting

    10–30μg protein samples were separated on 4–12% polyacrylamide Bis-Tris gels and transferred to polyvinylidene fluoride membranes. The blots were probed with METTL3-, METTL14-, HNRNPG- or GAPDH- specific primary antibody, followed by rabbit antigoat IgG-HRP or goat anti-rabbit IgG-HRP secondary antibody, and then visualized by enhanced chemoluminescence.

    Protein expression

    For expression of the N-terminal RNA recognition motif (N-RRM, residues 1–83) and C-terminal RNA binding domain (C-RBD, residues 334–391) of human HNRNPG protein, sequences encoding these HNRNPG domains were amplified by polymerase chain reaction (PCR) from human HeLa cDNA libraries and then subcloned into pGEX-6p-1 expression vectors using BamHI andXhoI restriction sites. PlasmidDNAwas transformed into Escherichia coli BL21-CodonPlus(DE3)-RP or BL21-CodonPlus(DE3)-RIL cells. The transformed bacteria were grown to saturation at 37oC, 200 rpm in Luria–Bertani Lennox medium with 100μg/ml ampicillin, then diluted 1:100, grown in the same culturemedium to an absorbance of∼0.6 at 600 nm, and induced with 1 mM isopropylβ-D-1-thiogalactoside (IPTG). The bacteria were grown an additional 16–22 h at 18oC, 200 rpm, then harvested and sonicated at 4◦C. GST-fusion proteins were isolated fromthe soluble lysate using glutathione-Sepharose beads and stored in 10 mM Tris-Cl (pH 7.4), 100 mM KCl, 2.5 mM MgCl2, 30% glycerol at −80oC.

    RNA oligos

    The following RNA oligos were synthesized2by Q.D. and purified by high-performance liquid chromatography (HPLC) and/or denaturing gel electrophoresis.

    The RNA oligos used:

    2,515-A: 5’- AAUGUGAAGGACUUUCGUAACGGAAGUAAUUCAA-Biotin;

    2,515-m6A: 5’- AAUGUGAAGGm6ACUUUCGUAACGGAAGUAAUUCAA-Biotin.

    RNA pull-down, gel shift and cross-linking

    The eluted protein samples were separated on 4–12% polyacrylamide Bis-Tris gelsand stained with SYPRO Ruby Protein Gel Stain according to themanufacturer’s instructions. Proteins in gel slices or in the entire pulled-down protein sample were digested with trypsin and identified using LC–MS/MS by the Donald Danforth Plant Science Center.

    The gel-purified 5’32Plabeled RNA oligos were refolded by heating at 90oC for 1 min, then at 30oC for 5 min. A total of 3μl nuclear extract and 6μl refolded RNA were incubated together at room temperature for 30 min, and then at 4oC for 2 h. Each sample was mixed with 1μl 50% glycerol, separated on an 8% polyacrylamide, 44.5 mMTris–borate (pH 8.3 at 25oC, pH 8.9 at 5oC), and 1 mM Na2EDTA (ethylenediaminetetraacetic acid) native gel at 5 ◦C, and visualized by phosphorimaging using the Personal Molecular Imager.

     

  • 动物实验

  • 不同实验动物依据体表面积的等效剂量转换表(数据来源于FDA指南)

    动物 A (mg/kg) = 动物 B (mg/kg)×动物 B的Km系数/动物 AKm系数


    例如,已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法:将88 mg/kg 乘以小鼠的Km系数(3),再除以大鼠的Km系数(6),得到该药物用于大鼠的等效剂量44 mg/kg。


  • 参考文献

    [1] Liu N, Zhou KI, Parisien M, Dai Q, Diatchenko L, Pan T. N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein. Nucleic Acids Res. 2017.
    more

    分子式
    C11H15N5O4
    分子量
    281.27
    CAS号
    1867-73-8
    储存方式
    ﹣20 ℃冷藏长期储存。冰袋运输
    溶剂(常温)
    DMSO
    50 mg/mL
    Water
    25 mg/mL
    Ethanol
    1 mg/mL

    体内溶解度

  • Clinical Trial Information ( data from http://clinicaltrials.gov )

    注:以上所有数据均来自公开文献,并不保证对所有实验均有效,数据仅供参考。

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