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25520
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Histone H3 Lysine Mutant-Specific Antibody Sampler Kit #25520

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Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 1
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) Antibody (upper) and Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower). As expected, the Histone H3 (K36M Mutant Specific) Antibody 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.
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 2
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) Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower). As expected, the Histone H3 (K36M Mutant Specific) Antibody 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.
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 3
Western blot analysis of extracts from various cell lines using Histone H3 (D1H2) XP® Rabbit mAb.
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 4
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Histone H3 (D1H2) XP® Rabbit mAb.
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 5
Confocal immunofluorescent analysis of HeLa cells using Histone H3 (D1H2) XP® Rabbit mAb (green) and β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red).
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 6
Flow cytometric analysis of HeLa cells using Histone H3 (D1H2) XP® Rabbit mAb (solid line) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 7
Western blot analysis of extracts from wild-type mES (mES/H3.3 WT), K9M mutant histone H3.3 knock-in mES (mES/H3.3 K9M), HeLa, and C2C12 cells, using Histone H3 (K9M Mutant Specific) (E2E9L) Rabbit mAb (upper) and Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower). As expected, Histone H3 (K9M Mutant Specific) (E2E9L) Rabbit mAb only detects the K9M 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.
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 8
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc-tagged wild-type histone H3 protein (Myc-Histone H3 WT; +) and Myc-tagged K9M mutant histone H3 protein (Myc-Histone H3 K9M; +), using Histone H3 (K9M Mutant Specific) (E2E9L) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and Histone H3 (D1H2) XP® Rabbit mAb #4499 (lower). As expected, Histone H3 (K9M Mutant Specific) (E2E9L) Rabbit mAb only detects the Myc-tagged K9M 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 K9M mutant histone H3 protein.
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 9
Immunoprecipitation of histone H3.3 K9M mutant protein from K9M mutant histone H3.3 knock-in mES cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Histone H3 (K9M Mutant Specific) (E2E9L) Rabbit mAb. Western blot analysis was performed using Histone H3 (K9M Mutant Specific) (E2E9L) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as the secondary antibody.
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 10
Confocal immunofluorescent analysis of mES cells containing either a knock-in K9M mutant histone H3.3 gene (left, positive) or a knock-in K36M mutant histone H3.3 gene (right, negative) using Histone H3 (K9M Mutant Specific) (E2E9L) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 11
Flow cytometric analysis of mES cells containing a knock-in K36M mutant histone H3.3 gene (blue) or a knock-in K9M mutant histone H3.3 gene (green) using Histone H3 (K9M Mutant Specific) (E2E9L) Rabbit mAb (solid lines) 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.
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 12
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 13
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).
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 14
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).
Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 15
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.
To Purchase # 25520T
Product # Size Price
25520T
1 Kit  (4 x 20 microliters) $ 336

Product Description

The Histone H3 Lysine Mutant-Specific Antibody Sampler Kit provides an economical means of detecting Lys (K) to Meth (M) cancer driver mutations in histone H3. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Histone H3 (K9M Mutant Specific) (E2E9L) Rabbit mAb recognizes endogenous levels of K9M mutant histone H3.1, H3.2, and H3.3 proteins. This antibody does not cross-react with wild-type histone H3.1, H3.2, or H3.3 proteins. Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb recognizes endogenous levels of K27M mutant histone H3.1, H3.2, and H3.3 proteins. This antibody may cross-react with wild-type histone H3.1, H3.2, and H3.3 when used at a high concentration. Careful titration of this antibody may be required to obtain optimal specificity. Histone H3 (K36M Mutant Specific) Antibody recognizes endogenous levels of K36M mutant histone H3.1, H3.2, and H3.3 proteins. This antibody may show slight cross-reactivity with wild-type histone H3.1, H3.2, or H3.3 when used at a high concentration. Careful titration of this antibody may be required to obtain optimal specificity. Histone H3 (D1H2) XP® Rabbit mAb detects endogenous levels of total Histone H3 protein, including isoforms H3.1, H3.2, and H3.3. This antibody also detects the Histone H3 variant CENP-A. This antibody does not cross-react with other core histones.

Source / Purification

Mutant-specific histone H3 monoclonal antibodies were produced by immunizing animals with synthetic peptides corresponding to K9M, K27M, or K36M mutant sequences of human histone H3.3 protein. Total histone H3 monoclonal antibody was produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of human histone H3 protein.

Background

Multiple exome sequencing analyses have uncovered a high frequency of histone H3 driver mutations in a number of different cancers, including diffuse intrinsic pontine glioma (DIPG), chondroblastoma, sarcomas, and HPV-negative head and neck squamous cell carcinoma. Previous studies have shown that lysine to methionine histone mutations in these cancers act as potent inhibitors of their respective lysine methyltransferases, resulting in gross alterations to the histone methylation landscape and deregulation of gene expression. In DIPG for example, the histone H3 K27M mutation is accompanied by a dramatic reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated tri-methylation of histone H3 lysine 27, changes in the distribution of PRC2 on the genome, and altered expression of genes associated with various cancer pathways (1-3). In chondrocytomas, the histone H3 K36M mutation functions to inhibit the WHSC1 (MMSET) and SETD2 histone methyltransferases, resulting in a reduction in the levels of histone H3 lysine 36 tri-methylation and deregulation of a number of cancer-associated genes (4). Similar to the H3K27M and H3K36M mutations, the histone H3 K9M mutation has been shown to inhibit the H3K9-directed histone methyltransferase G9a, resulting in reduced levels of histone H3 lysine 9 trimethylation (5). Given the widespread role of G9a in the regulation of gene expression, it is likely that this K9M mutation also plays a role in cancer.
  1. Chan, K.M. et al. (2013) Genes Dev 27, 985-90.
  2. Lewis, P.W. et al. (2013) Science 340, 857-61.
  3. Piunti, A. et al. (2017) Nat Med 23, 493-500.
  4. Fang, D. et al. (2016) Science 352, 1344-8.
  5. Jayaram, H. et al. (2016) Proc Natl Acad Sci U S A 113, 6182-7.

Pathways & Proteins

Explore pathways + proteins related to this product.

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