Antibodies, Proteins, Kits and Reagents for Life Science

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

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR18340] to Histone H3 (mutated K27M) - ChIP Grade
Suitable for: WB, Indirect ELISA, IHC-P, ICC/IF, IP, ChIP
Reacts with: Mouse, Human, Recombinant fragment

Product name

Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade
See all Histone H3 primary antibodies
Description

Rabbit monoclonal [EPR18340] to Histone H3 (mutated K27M) - ChIP Grade
Host species

Rabbit

Tested Applications & Species

Application	Species
ChIP	Human
ICC/IF	Human
IHC-P	Mouse Recombinant fragment
IP	Human
WB	Human Recombinant fragment

See all applications and species data

Immunogen

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR18340
Isotype

IgG
Research areas

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 ab213204 (red) (bottom panel, served as internal control) or 2 µg of rabbit normal IgG 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.

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol
Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

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

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 15 kDa
Observed band size: 18 kDa
why is the actual band size different from the predicted?

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

Exposure time: 6 seconds.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631) This image is courtesy of Dr Yusuke Tomita, Northwestern University.

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

Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631) at 1/1000 dilution + HEK-293 transfected with Histone H3.3 (K27M) expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 15 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Exposure time: 3 seconds

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.
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.
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) (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 (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
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 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. 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) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.
Western blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

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

Lane 1 : His-tagged recombinant histone H3 K27M protein
Lane 2 : His-tagged recombinant wild type histone H3 protein

Lysates/proteins at 0.01 µg per lane.

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

Predicted band size: 15 kDa
Observed band size: 15 kDa

Exposure time: 15 seconds

Blocking/Dilution buffer: 5% BSA/TBST.
Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (ab190631)

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