Revision 1

#69159Store at -20C

1 个试剂盒

(4 x 20 microliters)

Cell Signaling Technology

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3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
METTL3 (D2I6O) Rabbit mAb 96391 20 µl 70 kDa Rabbit IgG
METTL14 (D8K8W) Rabbit mAb 51104 20 µl 65 kDa Rabbit IgG
Virilizer (D4N8B) Rabbit mAb 88358 20 µl 210 kDa Rabbit IgG
WTAP Antibody 56501 20 µl 55 kDa Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The N6-mA Methyltransferase Antibody Sampler Kit provides an economical means of detecting N6-mA methyltransfearse proteins using control antibodies against METTL3, METTL14, WTAP, and Virilizer. The kit contains enough primary antibodies to perform at least two western blot experiments.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

Methyltransferase-like protein 3 (METTL3) and methyltransferase-like protein 14 (METTL14) are the two catalytic subunits of an N6-methyltransferase complex that methylates adenosine residues in RNA (1). Methylation of adenosine residues regulates mRNA splicing, processing, translation efficiency, editing and stability, in addition to regulating primary miRNA processing, and is critical for proper regulation of the circadian clock, embryonic stem cell self-renewal, immune tolerance, response to various stimuli, meiosis and mouse fertility (2,3). In this complex, METTL3 functions as the catalytic methyltransferase subunit and METTL14 functions as the target recognition subunit by binding to RNA (4). In addition, the Wilms' tumor 1-associating protein (WTAP) functions as a regulatory subunit and is required for accumulation of the complex to nuclear speckles, which are sites of RNA processing (5). Several studies suggest a role for this complex in cancer. METTL3 expression is elevated in lung adenocarcinoma where it promotes growth, survival, and invasion of human lung cancer cells (6). In addition, WTAP is overexpressed in a number of different cancers and positively regulates cell migration and invasion in glioblastoma and cholangiocarcinoma (7,8).

Virilizer is an additional protein subunit of the METTL3/METTL14 N6-methyltransferase complex and is required for methyltransferase activity (9). As part of the WTAP complex, Virilizer functions to regulate sex determination in Drosophila melanogaster through splicing of Sex-lethal (Sxl) mRNA transcripts (10,11).

  1. Liu, J. et al. (2014) Nat Chem Biol 10, 93-5.
  2. Yue, Y. et al. (2015) Genes Dev 29, 1343-55.
  3. Wu, R. et al. (2016) Mol Biotechnol 58, 450-9.
  4. Wang, X. et al. (2016) Nature 534, 575-8.
  5. Ping, X.L. et al. (2014) Cell Res 24, 177-89.
  6. Lin, S. et al. (2016) Mol Cell 62, 335-45.
  7. Jin, D.I. et al. (2012) Cancer Sci 103, 2102-9.
  8. Jo, H.J. et al. (2013) J Gastroenterol 48, 1271-82.
  9. Schwartz, S. et al. (2014) Cell Rep 8, 284-96.
  10. Hilfiker, A. et al. (1995) Development 121, 4017-26.
  11. SchüŁtt, C. et al. (1998) Development 125, 1501-7.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    限制使用

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    Revision 1
    #69159

    N6-mA Methyltransferase Antibody Sampler Kit

    N6-mA Methyltransferase Antibody Sampler Kit: Image 1 Expand Image

    Simple Western™ analysis of lysates (1 mg/mL) from NCCIT cells using METTL14 (D8K8W) Rabbit mAb #51104. 虚拟泳道式图像(左图)显示一抗稀释比例为 1:10 和 1:50 时的靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:10(蓝线)和 1:50(绿线)时沿毛细血管内分子量的化学发光结果。在还原条件下,使用 12-230 kDa 分离模块在 ProteinSimple(BioTechne 品牌)的 Jess™ Simple Western 仪器上进行该实验。

    N6-mA Methyltransferase Antibody Sampler Kit: Image 2 Expand Image
    使用 METTL14 (D8K8W) Rabbit mAb 对不同细胞系的提取物进行蛋白质印迹分析。
    N6-mA Methyltransferase Antibody Sampler Kit: Image 3 Expand Image
    使用 WTAP Antibody 对来自不同细胞系的提取物进行蛋白质印迹分析。
    N6-mA Methyltransferase Antibody Sampler Kit: Image 4 Expand Image
    一抗与靶标蛋白结合之后,与偶联 HRP 的二抗形成复合体。添加 LumiGLO®,在酶催化分解期间发光。
    N6-mA Methyltransferase Antibody Sampler Kit: Image 5 Expand Image
    使用 Virilizer (D4N8B) Rabbit mAb,对不同细胞系提取物进行蛋白质印迹分析。
    N6-mA Methyltransferase Antibody Sampler Kit: Image 6 Expand Image
    使用 METTL3 (D2I6O) Rabbit mAb,对来自不同细胞系的提取物进行蛋白质印迹分析。
    N6-mA Methyltransferase Antibody Sampler Kit: Image 7 Expand Image
    使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 对 mES 细胞的交联染色质与 Virilizer (D4N8B) Rabbit mAb 或 Normal Rabbit IgG #2729 进行染色质免疫沉淀。使用 SimpleChIP® Mouse Sox2 Exon 1 Primers #30180、mouse Klf2 intron 2 primers 和 SimpleChIP® Mouse MYT-1 Promoter Primers #8985,通过实时 PCR 对富集的 DNA 进行定量分析。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。
    N6-mA Methyltransferase Antibody Sampler Kit: Image 8 Expand Image
    对来自 293T 细胞提取物的 METTL3 进行免疫沉淀 。泳道 1 是10 % input,泳道 2 是 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900,而泳道 3 是 METTL3 (D2I6O) Rabbit mAb。使用 METTL3 (D2I6O) Rabbit mAb 和 Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 二抗进行蛋白质印迹分析。