Revision 1

#25520Store at -20C

1 Kit

(4 x 20 microliters)

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.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Histone H3 (K9M Mutant Specific) (E2E9L) Rabbit mAb 70414 20 µl 17 kDa Rabbit IgG
Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb 74829 20 µl 17 kDa Rabbit IgG
Histone H3 (K36M Mutant Specific) Antibody 26218 20 µl 17 kDa Rabbit 
Histone H3 (D1H2) XP® Rabbit mAb 4499 20 µl 17 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

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.

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 antibodies.

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.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a 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.

    使用に関する制限

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    Revision 1
    #25520

    Histone H3 Lysine Mutant-Specific Antibody Sampler Kit

    Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 1 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) 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 Expand Image
    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 3 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Histone H3 (D1H2) XP® Rabbit mAb.
    Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 4 Expand Image
    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 5 Expand Image
    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 6 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).
    Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 7 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) 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 8 Expand Image
    Immunohistochemical analysis of paraffin-embedded 4T1 syngeneic mammary tumor using Histone H3 (D1H2) XP® Rabbit mAb.
    Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 9 Expand Image
    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 10 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).
    Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 11 Expand Image
    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 12 Expand Image
    Immunohistochemical analysis of paraffin-embedded LL/2 syngeneic tumor using Histone H3 (D1H2) XP® Rabbit mAb.
    Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 13 Expand Image
    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 14 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.
    Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 15 Expand Image
    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 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse brain using Histone H3 (D1H2) XP® Rabbit mAb.
    Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 17 Expand Image
    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 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded rhesus monkey liver using Histone H3 (D1H2) XP® Rabbit mAb.
    Histone H3 Lysine Mutant-Specific Antibody Sampler Kit: Image 19 Expand Image
    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.