Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade - BSA and Azide free (AB240310)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling Histone H3 (K27 mutated to M) with ab190631 at 1/5000 (0.2 ug/ml) dilution, followed by ab190631 anti- H3(K27 mutated to M) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing nuclear staining in 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab190631).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade - BSA and Azide free (AB240310)

• IP

Unknown

Immunoprecipitation - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade - BSA and Azide free (AB240310)

Histone H3 (K27 mutated to M) was immunoprecipitated from 0.35 mg 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 : 293T (human embryonic kidney epithelial cell) cells transfected with myc-tagged H3(K27M) expression vector whole cell lysate 10ug

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

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab190631 in 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, azide, BSA and glycerol (ab190631).

All lanes:

Immunoprecipitation - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade (<a href='/en-us/products/primary-antibodies/histone-h3-mutated-k27m-antibody-epr18340-chip-grade-ab190631'>ab190631</a>)

Predicted band size: 15 kDa

Observed band size: 18 kDa

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• ChIP

Lab

ChIP - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade - BSA and Azide free (AB240310)

This data was developed using ab190631, the same antibody clone in a different buffer formulation.

Chromatin was prepared from 293T transfected with myc-His tagged Histone H3(K27 M) 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.

• Dot

Lab

Dot Blot - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade - BSA and Azide free (AB240310)

This data was developed using ab190631, the same antibody clone in a different buffer formulation.

Dot blot analysis of Histone H3 (mutated K27M) using ab190631 at 1 : 1000 followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (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 (<a href='/en-us/products/primary-antibodies/histone-h3-mutated-k27m-antibody-epr18340-chip-grade-ab190631'>ab190631</a>) 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

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Exposure time: 180s

• I-ELISA

Lab

Indirect ELISA - Anti-Histone H3 (mutated K27M) antibody [EPR18340] - ChIP Grade - BSA and Azide free (AB240310)

This data was developed using ab190631, the same antibody clone in a different buffer formulation.

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.