Revision 1
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, IF-IC, FC-FP

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

17

Source/Isotype:

Rabbit IgG

UniProt ID:

#P84243

Entrez-Gene Id:

3020

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunofluorescence (Immunocytochemistry) 1:1600
Flow Cytometry (Fixed/Permeabilized) 1:400 - 1:1600

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

Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb recognizes endogenous levels of K27M mutant histone H3.1, H3.2, and H3.3 proteins. The antibody may cross-react with wild-type histone H3.1, 3.2, and 3.3 when used at a high concentration. Careful titration of this antibody may be required to obtain optimal specificity.

Species Reactivity:

Human, Mouse

Species predicted to react based on 100% sequence homology

Rat, Xenopus, Pig

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to K27M mutant sequence of human histone H3.3 protein.

Background

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brainstem astrocyte tumor arising mostly in children, leading to a long-term survival rate of less than 10%. Multiple whole-genome sequencing studies of DIPG patients identified commonly occurring mutations in the H3F3A gene encoding histone H3.3. One of these mutations, a lysine to methionine amino acid substitution (K27M), is found in up to 78% of DIPGs and 22% of non-brainstem pediatric gliomas (1-3). This mutation is associated with poor prognosis, with a mean survival time of 0.73 years for patients with the K27M mutation versus 4.6 years for patients without the mutation (1-3). Expression of the K27M mutant histone H3 is accompanied by a dramatic reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated trimethylation of histone H3, changes in the distribution of PRC2 on the genome, and altered expression of genes associated with various cancer pathways (4-6).

  1. Wu, G. et al. (2012) Nat Genet 44, 251-3.
  2. Schwartzentruber, J. et al. (2012) Nature 482, 226-31.
  3. Khuong-Quang, D.A. et al. (2012) Acta Neuropathol 124, 439-47.
  4. Chan, K.M. et al. (2013) Genes Dev 27, 985-90.
  5. Lewis, P.W. et al. (2013) Science 340, 857-61.
  6. Piunti, A. et al. (2017) Nat Med 23, 493-500.

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 IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

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

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 1
#74829

Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb

Western Blotting Image 1: Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb Expand Image
使用 Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb(上图)、DYKDDDDK Tag (D6W5B) Rabbit mAb #14793(中图)和 Histone H3 (D1H2) XP® Rabbit mAb #4499(下图),对野生型小鼠星形胶质细胞和含敲入带 FLAG 标签的野生型组蛋白 H3.3 基因 (Flag-H3.3 WT) 或敲入带 FLAG 标签的 K27M 突变体组蛋白 H3.3 基因 (Flag-H3.3 K27M) 的小鼠星形胶质细胞的提取物进行蛋白质印迹分析。
Immunofluorescence Image 1: Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb Expand Image
使用 Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb(绿色)和 β-Actin (8H10D10) Mouse mAb #3700(红色),对含敲入带 FLAG 标签的 K27M 突变体组蛋白 H3.3 基因(左图,阳性)或敲入带 FLAG 标签的野生型组蛋白 H3.3 基因(中间,阴性)的小鼠星形胶质细胞和 HeLa 细胞(右图,阴性)进行共聚焦免疫荧光分析。
Flow Cytometry Image 1: Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb Expand Image
使用 Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)对包含敲入带 FLAG 标签的 K27M 突变型组蛋白 H3.3 基因(绿色)或敲入带 FLAG 标签的野生型组蛋白 H3.3 基因(蓝色)的小鼠星形胶质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。