Revision 7

#54062Store 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, ChIP, C&R

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

300

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q09472

Entrez-Gene Id:

2033

Product Information

Product Usage Information

For optimal ChIP 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.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:200
Chromatin IP 1:50
CUT&RUN 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

p300 (D2X6N) Rabbit mAb recognizes endogenous levels of total p300 protein. This antibody does not cross-react with CBP protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu733 of human p300 protein.

Background

CBP (CREB-binding protein) and p300 are highly conserved and functionally related transcriptional co-activators that associate with transcriptional regulators and signaling molecules, integrating multiple signal transduction pathways with the transcriptional machinery (1,2). CBP/p300 also contain histone acetyltransferase (HAT) activity, allowing them to acetylate histones and other proteins (2). Phosphorylation of p300 at Ser89 by PKC represses its transcriptional activity, and phosphorylation at the same site by AMPK disrupts the association of p300 with nuclear receptors (3,4). Ser1834 phosphorylation of p300 by Akt disrupts its association with C/EBPβ (5). Growth factors induce phosphorylation of CBP at Ser437, which is required for CBP recruitment to the transcription complex (6). CaM kinase IV phosphorylates CBP at Ser302, which is required for CBP-dependent transcriptional activation in the CNS (7). The role of acetylation of CBP/p300 is of particular interest (2,8). Acetylation of p300 at Lys1499 has been demonstrated to enhance its HAT activity and affect a wide variety of signaling events (9).

  1. Goodman, R.H. and Smolik, S. (2000) Genes Dev 14, 1553-77.
  2. Chan, H.M. and La Thangue, N.B. (2001) J. Cell Sci. 114, 2363-2373.
  3. Yuan, L.W. and Gambee, J.E. (2000) J. Biol. Chem. 275, 40946-40951.
  4. Yang, W. et al. (2001) J. Biol. Chem. 276, 38341-38344.
  5. Guo, S. et al. (2001) J. Biol. Chem. 276, 8516-8523.
  6. Zanger, K. et al. (2001) Mol. Cell 7, 551-558.
  7. Impey, S. et al. (2002) Neuron 34, 235-244.
  8. Yuan, L.W. and Giordano, A. (2002) Oncogene 21, 2253-2260.
  9. Thompson, P.R. et al. (2004) Nat. Struct. Mol. Biol. 11, 308-315.

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 nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation ChIP: Chromatin IP C&R: CUT&RUN

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.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
XP is a registered 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 7
#54062

p300 (D2X6N) Rabbit mAb

Western Blotting Image 1: p300 (D2X6N) Rabbit mAb Expand Image
使用 p300 (D2X6N) Rabbit mAb(上图)和 α-Actinin (D6F6) XP® Rabbit mAb #6487(下图)对 293T 和 HCT-15 细胞的提取物进行蛋白质印迹分析。和预期一样,HCT-15 细胞中 p300 表达呈阴性。
No image available
Chromatin Immunoprecipitation Image 1: p300 (D2X6N) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对 K-562 细胞中提取的交联染色质,在加入 p300 (D2X6N) 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 FDPS 基因内的结合作用。欲知其他 ChIP-seq 情况,请下载产品说明书。
Chromatin Immunoprecipitation Image 2: p300 (D2X6N) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对 K-562 细胞中提取的交联染色质,在加入 p300 (D2X6N) 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 1(上图)内的结合,包括 FDPS 基因(下图)。
Chromatin Immunoprecipitation Image 3: p300 (D2X6N) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 对 K-562 细胞中提取的交联染色质,在加入 p300 (D2X6N) Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀。使用 human STAT3 promoter primers, SimpleChIP® Human HNRNPA0 Promoter Primers #83602 和 SimpleChIP® Human MYT-1 Exon 1 Primers #4493,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。
CUT and RUN Image 1: p300 (D2X6N) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 K-562 细胞和 p300 (D2X6N) Rabbit mAb 进行 CUT&RUN 检测。‚使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示了 p300 的已知靶标基因 HNRNPA0 的结合(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
CUT and RUN Image 2: p300 (D2X6N) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 K-562 细胞和 p300 (D2X6N) Rabbit mAb 进行 CUT&RUN 检测。‚使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示在染色体 5(上图)内的结合,包括 p300 的已知靶标基因 HNRNPA0(下图)(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
CUT and RUN Image 3: p300 (D2X6N) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对€‚ K-562 细胞和 p300 (D2X6N) Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN 检测。使用 SimpleChIP® Human HNRNPA0 Promoter Primers #83602 和 SimpleChIP® Human α Satellite Repeat Primers #4486,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。