Revision 3

#84406Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IF-IC, FC-FP, ChIP, ChIP-seq

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

62

Source/Isotype:

Rabbit IgG

UniProt ID:

#P04198

Entrez-Gene Id:

4613

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:200
Immunofluorescence (Immunocytochemistry) 1:100 - 1:400
Flow Cytometry (Fixed/Permeabilized) 1:200 - 1:800
Chromatin IP 1:50
Chromatin IP-seq 1:50

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.

Specificity / Sensitivity

N-Myc (D1V2A) Rabbit mAb recognizes endogenous levels of total N-Myc protein.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro335 of human N-Myc protein.

Background

Members of the Myc/Max/Mad network function as transcriptional regulators with roles in various aspects of cell behavior, including proliferation, differentiation, and apoptosis (1). These proteins share a common basic-helix-loop-helix leucine zipper (bHLH-ZIP) motif required for dimerization and DNA-binding. Max was originally discovered based on its ability to associate with c-Myc and found to be required for the ability of Myc to bind DNA and activate transcription (2). Subsequently, Max has been viewed as a central component of the transcriptional network, forming homodimers as well as heterodimers with other members of the Myc and Mad families (1). The association between Max and either Myc or Mad can have opposing effects on transcriptional regulation and cell behavior (1). The Mad family consists of four related proteins; Mad1, Mad2 (Mxi1), Mad3, and Mad4, and the more distantly related members of the bHLH-ZIP family, Mnt and Mga. Like Myc, the Mad proteins are tightly regulated with short half-lives. In general, Mad family members interfere with Myc-mediated processes, such as proliferation, transformation, and prevention of apoptosis by inhibiting transcription (3,4).

In humans the Myc family consists of 5 genes: c-Myc, N-Myc, L-Myc, R-Myc, and B-Myc. While c-Myc is expressed in many proliferating cells, N-Myc expression is very restricted, with highest levels in during embryonic development and then in the adult during B-cell development. These expression patterns and results from targeted deletion of N-Myc suggest that N-Myc plays an important role in tissue development and differentiation (5). In addition, amplification or overexpression of N-Myc has been found in human neuroblastomas and is associated with rapid progression and poor prognosis (6,7).

  1. Baudino, T.A. and Cleveland, J.L. (2001) Mol Cell Biol 21, 691-702.
  2. Blackwood, E.M. and Eisenman, R.N. (1991) Science 251, 1211-7.
  3. Henriksson, M. and Lüscher, B. (1996) Adv Cancer Res 68, 109-82.
  4. Grandori, C. et al. (2000) Annu Rev Cell Dev Biol 16, 653-99.
  5. Brodeur, G.M. et al. (1984) Science 224, 1121-1124.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
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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专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 3
#84406

N-Myc (D1V2A) Rabbit mAb

Western Blotting Image 1: N-Myc (D1V2A) Rabbit mAb Expand Image
使用 N-Myc (D1V2A) Rabbit mAb(上图)或 β-Actin (D6A8) Rabbit mAb #8457(下图)对不同细胞系提取物进行蛋白质印迹分析。
Immunoprecipitation Image 1: N-Myc (D1V2A) Rabbit mAb Expand Image
对 IMR-32 细胞提取物 N-Myc 进行免疫沉淀。泳道 1 为10% input,泳道2是用 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900沉淀的,而泳道3为 N-Myc (D1V2A) Rabbit mAb。使用 N-Myc (D1V2A) Rabbit mAb 进行蛋白质印迹操作。证实特异性二抗可用于避免与 IgG 的交叉反应。
Immunofluorescence Image 1: N-Myc (D1V2A) Rabbit mAb Expand Image
使用 N-Myc (D1V2A) Rabbit mAb (绿色)对 IMR-32 (阳性,左)和 HeLa (阴性,右)细胞进行共聚焦免疫荧光分析。肌动蛋白丝已用 DyLight 554 Phalloidin #13054(红色)进行标记。
Flow Cytometry Image 1: N-Myc (D1V2A) Rabbit mAb Expand Image
使用 N-Myc (D1V2A) Rabbit mAb 对 HeLa 细胞(蓝色)和 IMR32 细胞(绿色)进行流式细胞分析。Anti-rabbit IgG (H+L)、F(ab')2 Fragments (Alexa Fluor® 488 Conjugate) #4412 作为二抗。。
Chromatin Immunoprecipitation Image 1: N-Myc (D1V2A) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对 IMR-32 细胞中提取的交联染色质,在加入 N-Myc (D4B2Y) Rabbit mAb #51705 或 N-Myc (D1V2A) Rabbit mAb 后进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。图片中显示在 MIR17HG 内的结合,而 MIR17HG 是知名的 N-Myc 靶标基因(请参阅含有 ChIP-qPCR 数据的附图)。如需了解其他 ChIP-seq 情况,请下载产品说明书。
Chromatin Immunoprecipitation Image 2: N-Myc (D1V2A) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对 IMR-32 细胞中提取的交联染色质,在加入 N-Myc (D4B2Y) Rabbit mAb #51705 或 N-Myc (D1V2A) Rabbit mAb 后进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在染色体 13(上图)内的结合,包括 N-Myc 的已知靶标基因 MIR17HG(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: N-Myc (D1V2A) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对 IMR-32 细胞中提取的交联染色质,在加入 N-Myc (D1V2A) Rabbit mAb 或 Normal Rabbit IgG #2729 后进行染色质免疫沉淀。采用人 MIR17HG intron 1 primers、SimpleChIP® Human MDM2 Intron 2 Primers #90678和 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 对富集的 DNA 进行量化。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。