Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade

10 product images

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  ChIP - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  Chromatin was prepared from HEK-293T transfected with myc-His tagged Histone H3 (K27M) and Histone H3 WT cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
  The ChIP was performed with 25 μg of chromatin, 2 μg of ab190631 (red), 2 μg of Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204 (red) (bottom panel, served as internal control) or 2 μg of rabbit normal IgG Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 (gray) and 20 μl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
  

  Primers and probes are located in the first kb of the transcribed region.

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  Immunoprecipitation - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  Histone H3 (K27 mutated to M) was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) cells transfected with myc-tagged H3(K27M) expression vector whole cell lysate 10ug with ab190631 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab190631 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
  

  Lane 1: HEK-293T (human embryonic kidney epithelial cell) cells transfected with myc-tagged H3(K27M) expression vector whole cell lysate 10ug

  Lane 2: ab190631 IP in HEK-293T cells transfected with myc-tagged H3(K27M) expression vector whole cell lysate

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab190631 in HEK-293T cells transfected with myc-tagged H3(K27M) expression vector whole cell lysate

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  Exposure time: 5 seconds

  All lanes: Immunoprecipitation - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  Predicted band size: 15 kDa

  Observed band size: 18 kDa

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  Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  Exposure time: 6 seconds.

  All lanes: Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631) at 1/1000 dilution

  Lane 1: HEK-293 transfected with Histone H3.1 (K27M) expression vector containing a myc-His-tag, whole cell lysate at 10 µg

  Lane 2: HEK-293 transfected with Histone H3(WT) expression vector containing a myc-His-tag, whole cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 15 kDa

  Observed band size: 18 kDa

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  This image is courtesy of Dr Yusuke Tomita, Northwestern University.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of tissue sections from mouse brain tumor with and without H3K27M mutation labeling Histone H3 (mutated K27M) with ab190631 at 1/500 dilution (1 hour at 36°C in 2% BSA in PBS).Tissue was fixed with 20% Neutral Buffer Formalin for 48 hours, then changed to 70% ethanol for a week. Paraffin-embedded tissue was cut in 5 micrometer thickness with microtome. Antigen retrieval was done with Cell-Conditioning 1 (Roche) at 95 degree. A HRP conjugated monoclonal antibody (DISCOVERY UltraMap anti-Rb HRP (RUO)) was used as the secondary antibody.

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  Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

  All lanes: Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631) at 1/1000 dilution

  All lanes: HEK-293 transfected with Histone H3.3 (K27M) expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 15 kDa

  Observed band size: 18 kDa

  Exposure time: 3s

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  Indirect ELISA - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  ELISA using ab190631 at varying antibody concentrations and antigen concentration at 1000 ng/mL. An Alkaline Phosphatase-conjugated Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.
  

  The blue line indicates binding to the Histone H3 (mutated K27M) peptide. Binding to the following peptides was not seen:

  Histone H3 WT,
  Histone H3 (mono methyl K27),
  Histone H3 (di methyl K27),
  Histone H3 (tri methyl K27).

  This indicates the specificity of ab190631 for mutated K27M of Histone H3.

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human embryonic kidney epithelial cell) cells labeling Histone H3 (K27 mutated to M) with ab190631 at 1/5000 (0.2 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing nuclear staining in HEK-293T cells transfected with myc-tagged H3 (K27 mutated to M) expression vector is observed. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
  

  Secondary antibody only control: Secondary antibody is ab190631 anti- H3(K27 mutated to M) Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

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  Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  Blocking/Dilution buffer: 5% BSA/TBST.

  All lanes: Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631) at 1/1000 dilution

  Lane 1: His-tagged recombinant histone H3 K27M protein at 0.01 µg

  Lane 2: His-tagged recombinant wild type histone H3 protein at 0.01 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 15 kDa

  Observed band size: 15 kDa

  Exposure time: 15s

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of tissue sections from(A) HEK-293T transfected with Histone H3 (mutated K27M) expression vector containing a his tag. (B) HEK-293T transfected with wildtype Histone H3 expression vector containing a his tag. (C) HEK-293T transfected with empty vector containing a his tag, labeling Histone H3 (mutated K27M) with ab190631 at 1/1000 dilution and ready to use secondary LeicaDS9800 (Bond™ Polymer Refine Detection) counterstained with Hematoxylin.
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
  
  Positive staining on HEK-293T transfected with Histone H3 (mutated K27M) expression vector cell pellets (image A), no staining on HEK-293T transfected with wildtype Histone H3 expression vector cell pellets (Image B) or empty vector cell pellets (Image C).
  The section was incubated with ab190631 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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  Dot Blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

  Dot blot analysis of Histone H3 (mutated K27M) using ab190631 at 1:1000 followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1:100,000 dilution.

  Lane 1: Histone H3 (K27 mutated to M) peptide
  
  Lane 2: Histone H3 unmodified peptide
  
  Lane 3: Histone H3 (tri methyl K18 + tri methyl K36) peptide

  All lanes: Dot Blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631) at 1/1000 dilution

  Lane 1: H3 (K27 mutated to M) aa22-32

  Lane 2: H3 H3 unmod(1-33)

  Lane 3: H3 K18me3 + K36me3 15-39

  Secondary

  All lanes: Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Exposure time: 180s