Histone H3 (mutated K27 M) Recombinant Rabbit Monoclonal Antibody [JE46-51]
cat.: HA722200
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse
Applications: WB, IF-Cell, IHC-P, ChIP
Clonality: Monoclonal
Clone number: JE46-51
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 15 kDa
Isotype: IgG
Immunogen: Synthetic peptide within Human Histone H3.3 aa 1-50 (mutated K27 M).
Positive control: 293T-OE-Histone H3 (mutated K27 M) cell lysate, HeLa cells transfected with Histone H3 (mutated K27 M).
Subcellular location: Nucleus, Chromosome.
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
  ChIP
1:1,000-1:5,000
1:1,000
1:1,000
Use 0.5~2 μg for 25 μg of chromatin.
Uniprot #: SwissProt: P68431 Human
Alternative names: H3 histone family member E pseudogene H3 histone family, member A H3/A H31_HUMAN H3F3 H3FA Hist1h3a HIST1H3B HIST1H3C HIST1H3D HIST1H3E HIST1H3F HIST1H3G HIST1H3H HIST1H3I HIST1H3J HIST3H3 histone 1, H3a Histone cluster 1, H3a Histone H3 3 pseudogene Histone H3.1 Histone H3/a Histone H3/b Histone H3/c Histone H3/d Histone H3/f Histone H3/h Histone H3/i Histone H3/j Histone H3/k Histone H3/l
Images
HA722200_1.jpg Fig1: Western blot analysis of Histone H3 (mutated K27 M) on different lysates with Rabbit anti-Histone H3 (mutated K27 M) antibody (HA722200) at 1/1,000 dilution.

Lane 1: 293T-OE-Histone H3 (mutated K27 M) cell lysate
Lane 2: 293T-OE-Histone H3 cell lysate (negative)

Lysates/proteins at 10 µg/Lane.

Predicted band size: 15 kDa
Observed band size: 18 kDa

Exposure time: 2 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722200) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HA722200_2.jpg Fig2: Western blot analysis of Histone H3 (mutated K27 M) on different lysates with Rabbit anti-Histone H3 (mutated K27 M) antibody (HA722200) at 1/2,000 dilution.

Lane 1: HeLa cell lysate (negative)
Lane 2: Raji cell lysate (negative)
Lane 3: 293T cell lysate (negative)
Lane 4: HT-29 cell lysate (negative)
Lane 5: Jurkat cell lysate (negative)

Lysates/proteins at 30 µg/Lane.

Exposure time: 3 minutes; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722200) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HA722200_3.jpg Fig3: Immunocytochemistry analysis of HeLa cells transfected with Histone H3 / Histone H3 (mutated K27 M) labeling Histone H3 (mutated K27 M) with Rabbit anti-Histone H3 (mutated K27 M) antibody (HA722200) at 1/1,000 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Histone H3 (mutated K27 M) antibody (HA722200) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HA722200_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded 293T cells transfected with Histone H3 / Histone H3 (mutated K27 M) with Rabbit anti-Histone H3 (mutated K27 M) antibody (HA722200) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA722200) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HA722200_5.jpg Fig5: Chromatin immunoprecipitations were performed with cross-linked chromatin from 293T cells transfected with Histone H3 (mutated K27 M) cells with Histone H3 (mutated K27 M) (HA722200) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
HA722200_6.jpg Fig6: Chromatin immunoprecipitations were performed with cross-linked chromatin from 293T cells transfected with Histone H3 cells (negative) with Histone H3 (mutated K27 M) (HA722200) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.