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.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

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
Western blot analyis of extracts from wild-type mouse astrocytes, and mouse astrocytes containing either a knock-in FLAG-tagged wild-type histone H3.3 gene (Flag-H3.3 WT) or a knock-in FLAG-tagged K27M mutant histone H3.3 gene (Flag-H3.3 K27M), using Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb (upper), DYKDDDDK Tag (D6W5B) Rabbit mAb #14793 (middle), and Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower).
Immunofluorescence Image 1: Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of mouse astrocytes, containing either a knock-in FLAG-tagged K27M mutant histone H3.3 gene (left, positive) or a knock-in FLAG-tagged wild-type histone H3.3 gene (center, negative), and HeLa cells (right, negative), using Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red).
Flow Cytometry Image 1: Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb Expand Image
Flow cytometric analysis of mouse astrocytes, containing either a knock-in FLAG-tagged K27M mutant histone H3.3 gene (green) or a knock-in FLAG-tagged wild-type histone H3.3 gene (blue), using Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb (solid lines) or or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.