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, ChIP

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

17

Source/Isotype:

Rabbit IgG

UniProt ID:

#P84243

Entrez-Gene Id:

3020

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.
Application Dilution
Western Blotting 1:1000
Immunofluorescence (Immunocytochemistry) 1:1600 - 1:6400
Chromatin IP 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

Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb recognizes endogenous levels of K36M mutant histone H3.1, H3.2, and H3.3 proteins. The antibody may show slight cross-reactivity with wild-type histone H3.1, 3.2, or 3.3 when used at a high concentration. Careful titration of this antibody may be required to obtain optimal specificity. This antibody may detect a band of unknown origin at 50 kDa.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to K36M mutant sequence of human histone H3.3 protein.

Background

Chondroblastoma is a rare type of benign tumor that is found at the rounded ends of the long bones in the arms and legs. More than 90% of chondroblastomas have been found to contain a heterozygous mutation in the H3F3A gene encoding the histone variant H3.3 (1). This mutation, a lysine to methionine amino acid substitution in codon 36 (K36M), inhibits at least two histone H3 lysine 36 methyltransferases, WHSC1 (MMSET) and SETD2, resulting in reduced global levels of histone H3 lysine 36 methylation (1). Chondrocytes containing the histone H3 K36M mutation exhibit several hallmarks of cancer cells, including increased ability to form colonies, resistance to apoptosis, and defects in differentiation. Reduction of global methylation levels in chondrocytes, resulting from the K36M mutation, contributes to tumorigenesis by altering the expression of cancer-associated genes. The histone H3 K36M mutation is also found to promote sarcomagenesis by impairing the differentiation of mesenchymal progenitor cells, resulting in undifferentiated sarcomas (2). The K36M mutation alters the histone methylation landscape, resulting in a genome-wide gain in histone H3 lysine 27 methylation, redistribution of polycomb repressive complex 1, and derepression of its target genes known to block mesenchymal differentiation. Finally, the histone H3 K36M mutation is also found in 13% of HPV-negative head and neck squamous cell carcinomas, again contributing to tumorigenesis by altering global methylation levels of histone H3 lysine 36 (3).

  1. Fang, D. et al. (2016) Science 352, 1344-8.
  2. Lu, C. et al. (2016) Science 352, 844-9.
  3. Papillon-Cavanagh, S. et al. (2017) Nat Genet 49, 180-5.

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 IF-IC: Immunofluorescence (Immunocytochemistry) ChIP: Chromatin IP

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.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 1
#98399

Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb

Western Blotting Image 1: Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb Expand Image
Western blot analysis of extracts from wild-type mES cells (mES/H3.3 WT) and K36M mutant histone H3.3 knock-in mES cells (mES/H3.3 K36M), using Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb (upper) or Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower). As expected, the Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb only detects the K36M mutant histone H3.3 protein and not the wild-type H3.3 protein. Cell lines were generously provided by Dr. Konrad Hochedlinger at Massachusetts General Hospital.
Western Blotting Image 2: Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected (+) with Myc-tagged wild-type histone H3 (Myc-Histone H3 WT) or Myc-tagged K36M mutant histone H3 (Myc-Histone H3 K36M), using Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), or Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower). As expected, the Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb only detects the Myc-tagged K36M mutant histone H3 protein and not the Myc-tagged wild-type histone H3 or endogenous wild-type histone H3 proteins. This antibody does detect endogenous levels of K36M mutant histone H3 protein.
Immunofluorescence Image 1: Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of mES cells containing either a knock-in K36M mutant histone H3.3 gene (left, positive) or wild-type histone H3.3 gene (right, negative) using Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb (green), DyLight 650 Phalloidin #12956 (red), and DAPI #4083 (blue).
Immunofluorescence Image 2: Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HCT 116 cells, either transfected with H3.3 K36M protein (left) or transfected with H3.3 wild-type protein (right), using Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb (green), DyLight 650 Phalloidin #12956 (red), and DAPI #4083 (blue).
Chromatin Immunoprecipitation Image 1: Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from mES/H3.3 wild-type cells (left panel) and mES/H3.3 K36M cells (right panel), and either Histone H3 (K36M Mutant Specific) (E8N1L) Rabbit mAb or Normal Rabbit IgG #2729, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using mouse Dguok promoter primers and SimpleChIP® Mouse Intracisternal A-Particle (IAP) LTR Primers #85916. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.