Revision 6
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, W-S, IP, IF-IC, FC-FP, ChIP, ChIP-seq

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

17

Source/Isotype:

Rabbit IgG

UniProt ID:

#P68431

Entrez-Gene Id:

8350

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
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:50
Immunofluorescence (Immunocytochemistry) 1:12800 - 1:25600
Flow Cytometry (Fixed/Permeabilized) 1:1600 - 1:6400
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

Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb recognizes endogenous levels of Histone H3 protein only when acetylated at Lys14. This antibody does not cross react with histone H3 acetylated at Lys9, 18, 27, or 56.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology

Hamster, D. melanogaster, Xenopus, Zebrafish, Pig, S. cerevisiae, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding acetylated Lys14 of human Histone H3 protein.

Background

The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (1,2). Histone acetylation occurs mainly on the amino-terminal tail domains of histones H2A (Lys5), H2B (Lys5, 12, 15, and 20), H3 (Lys9, 14, 18, 23, 27, 36, and 56), and H4 (Lys5, 8, 12, and 16) and is important for the regulation of histone deposition, transcriptional activation, DNA replication, recombination, and DNA repair (1-3). Hyper-acetylation of the histone tails neutralizes the positive charge of these domains and is believed to weaken histone-DNA and nucleosome-nucleosome interactions, thereby destabilizing chromatin structure and increasing the accessibility of DNA to various DNA-binding proteins (4,5). In addition, acetylation of specific lysine residues creates docking sites for a protein module called the bromodomain, which binds to acetylated lysine residues (6). Many transcription and chromatin regulatory proteins contain bromodomains and may be recruited to gene promoters, in part, through binding of acetylated histone tails. Histone acetylation is mediated by histone acetyltransferases (HATs), such as CBP/p300, GCN5L2, PCAF, and Tip60, which are recruited to genes by DNA-bound protein factors to facilitate transcriptional activation (3). Deacetylation, which is mediated by histone deacetylases (HDAC and sirtuin proteins), reverses the effects of acetylation and generally facilitates transcriptional repression (7,8).

  1. Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.
  2. Jaskelioff, M. and Peterson, C.L. (2003) Nat Cell Biol 5, 395-9.
  3. Roth, S.Y. et al. (2001) Annu Rev Biochem 70, 81-120.
  4. Workman, J.L. and Kingston, R.E. (1998) Annu Rev Biochem 67, 545-79.
  5. Hansen, J.C. et al. (1998) Biochemistry 37, 17637-41.
  6. Yang, X.J. (2004) Bioessays 26, 1076-87.
  7. Haberland, M. et al. (2009) Nat Rev Genet 10, 32-42.
  8. Haigis, M.C. and Sinclair, D.A. (2010) Annu Rev Pathol 5, 253-95.

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 W-S: Simple Western™ 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.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

除非 CST 的合法授书代表以书面形式书行明确同意,否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件,除非书 CST 的合法授书代表以书面形式书独接受, 否书均被拒书,并且无效。

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 6
#7627

Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb

Western Blotting Image 1: Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb Expand Image
使用 Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb(上)或 Histone H3 (D1H2) XP® Rabbit mAb #4499(下)时,对未经 (-) 或经 Trichostatin A (TSA) #9950(1 μM,18 小时)处理 (+) 的 HeLa 细胞、C2C12 细胞和 COS-7 细胞提取物进行蛋白质印迹分析。
Western Blotting Image 1: Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb Expand Image
使用 Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb #7627 对经过 Trichostatin A(1uM,过夜 18 小时)处理的 HeLa 细胞的裂解物 (1 mg/mL) 进行 Simple Western™ 分析。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。
No image available
Immunofluorescence Image 1: Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb Expand Image
使用 Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb(绿色)对未处理(左)或经 Trichostatin A (TSA) #9950 (1 uM 18小时;右)处理的 HeLa 细胞进行共聚焦免疫荧光分析。肌动蛋白微丝由 DyLight 554 Phalloidin #13054(红色)标记。
Flow Cytometry Image 1: Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb Expand Image
使用 Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb #7627(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线),对未经处理(蓝色)或已经过 Trichostatin A #9950(1 μM,4 小时;绿色)处理的 HeLa 细胞进行流式细胞分析。Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 用作二抗。
Chromatin Immunoprecipitation Image 1: Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb Expand Image

使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对 HeLa 细胞中提取的交联染色质,在加入 Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb 后,进行染色质免疫沉淀。使用 SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina Systems #56795 制备 DNA 库。结果图显示在 VMP1 基因内的结合作用。

Chromatin Immunoprecipitation Image 2: Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb Expand Image

使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对 HeLa 细胞中提取的交联染色质,在加入 Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb 后,进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示在染色体 17(上图)内的结合,包括 VMP1 基因(下图)。

Chromatin Immunoprecipitation Image 3: Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对 HeLa 细胞中提取的交联染色质,在加入 Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀。使用 SimpleChIP® Human GAPDH Intron 2 Primers #4478、SimpleChIP® Human RPL30 Exon 3 Primers #7014、SimpleChIP® Human MyoD1 Exon 1 Primers #4490 和 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 对富集的 DNA进行定量。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。