Anti-Histone H3 (mutated K36M) antibody [EPR23614-91]

9 product images

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  Immunoprecipitation - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384)

  Histone H3.3 (mutated K36 M) was immunoprecipitated from 0.35 mg HEK-293 transfected with Histone H3.3 K36M (mutate) expression vector containing a myc-His-tag^®, whole cell lysate with ab256384 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256384 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-293 transfected with Histone H3.3 K36M (mutate) expression vector containing a myc-His-tag®, whole cell lysate 10 ug

  Lane 2: ab256384 IP in HEK-293 transfected with Histone H3.3 K36M (mutate) expression vector containing a myc-His-tag®, whole cell lysate

  Lane 3:Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab256384 in HEK-293 transfected with Histone H3.3 K36M (mutate) expression vector containing a myc-His-tag^®, whole cell lysate

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

  Exposure time: 10 seconds

  All lanes: Immunoprecipitation - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384)

  Predicted band size: 15 kDa

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  Western blot - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384)

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

  Exposure time: 26 seconds

  All lanes: Western blot - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384) at 1/1000 dilution

  Lane 1: HEK-293 transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

  Lane 2: HEK-293 transfected with Histone H3.3 K36M (mutate) 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: 20 kDa

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  Flow Cytometry (Intracellular) - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384)

  Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T transfected with myc tagged Histone H3 construct (Left) or myc tagged Histone H3 K36M construct (Right) cells labelling Histone H3 (mutated K36 M) with ab256384 at 1/500 compared with a isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 was used as the secondary antibody.
  

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  Indirect ELISA - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384)

  Indirect ELISA using ab256384 at varying antibody concentrations and antigen concentration at 100 ng/mL. An Alkaline Phosphatase-conjugated Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.
  

  Binding was seen for Histone H3 (mutated K36M) peptide. Binding to the following peptides was not seen:

  Histone H3 WT,
  Histone H3 (mono methyl K36),
  Histone H3 (di methyl K36),
  Histone H3 (tri methyl K36).

  This indicates the specificity of ab256384 for mutated K36M of Histone H3.

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293 cells labelling Histone H3 (mutated K36 M) with ab256384 at 1/50 dilution, followed by a secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HEK-293 cell line transfected with myc-tagged Histone H3 K36M expression vector. 2233S Myc-Tag (9B11) Mouse mAb (Alexa Fluor® 647 Conjugate) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384)

  Immunohistochemical analysis of paraffin-embedded human giant cell tumor of bone tissue labeling Histone H3 (mutated K36 M) with ab256384 at 1/4000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Negative control: No staining in human giant cell tumor of bone (PMID: 29757500). Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

  Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0)

• 

  Western blot - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384)

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

  All lanes: Western blot - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384) at 1/1000 dilution

  Lane 1: HEK-293 transfected with Histone H3.1 (WT) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

  Lane 2: HEK-293 transfected with Histone H3.1 K36M (mutant) expression vector containing a myc-His-tag®, whole cell lysate at 20 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: 19 kDa

  Exposure time: 62s

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384)

  Immunohistochemical analysis of paraffin-embedded human chondroblastoma tissue labeling Histone H3 (mutated K36 M) with ab256384 at 1/4000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in human chondroblastoma (PMID: 29757500). Counterstained with Hematoxylin.
  

  Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

  Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0)

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  ChIC/CUT&RUN sequencing - Anti-Histone H3 (mutated K36M) antibody [EPR23614-91] (ab256384)

  ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 293T cells transfected with a H3.3K36M (Mutant) or H3.3 (WT) plasmid and 3µg of ab256384 [EPR23614-91]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.
  
  Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.
  
  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.