Histone H3 (tri methyl K4) Recombinant Rabbit Monoclonal Antibody [PSH25-68]

Western blot analysis of Histone H3 (tri methyl K4) on different lysates with Rabbit anti-Histone H3 (tri methyl K4) antibody (HA724555) at 1/2,000 dilution.

Lane 1: HeLa (Human cervical adenocarcinoma cell) cell lysate
Lane 2: U-2 OS (Human osteosarcoma cell) cell lysate
Lane 3: NIH/3T3 (Mouse fibroblast) cell lysate
Lane 4: C2C12 (Mouse myoblast) cell lysate
Lane 5: C6 (Rat glioma cell) cell lysate
Lane 6: PC-12 (Rat pheochromocytoma cell (undifferentiated)) cell lysate
Lane 7: COS-1 (African green monkey kidney fibroblast) cell lysate

Lysates/proteins at 20 µg/Lane.
Exposure time: 1 minute 30 seconds; ECL: K1801

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA724555, 1/2,000 in primary antibody dilution buffer (K1803), overnight at 4 °C
Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

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

Application: Immunocytochemistry (IF-cell)

Species: Human
Sample: HeLa (Human cervical adenocarcinoma cell)

Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.
Permeabilization: 0.1% Triton X-100, 15 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 1 hour at room temperature.
Antibody dilution buffer: 1% BSA in PBST.

Primary antibody: HA724555, 1/500, overnight at 4°C.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 45 minutes at room temperature.
Counterstain: Beta tubulin (HA601187, Red), 1/100, overnight at 4℃. The nuclear counterstain was DAPI (Blue).

Application: Immunocytochemistry (IF-cell)

Species: Mouse
Sample: NIH/3T3 (Mouse fibroblast)

Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.
Permeabilization: 0.1% Triton X-100, 15 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 1 hour at room temperature.
Antibody dilution buffer: 1% BSA in PBST.

Primary antibody: HA724555, 1/500, overnight at 4°C.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 45 minutes at room temperature.
Counterstain: Beta tubulin (HA601187, Red), 1/100, overnight at 4℃. The nuclear counterstain was DAPI (Blue).

Application: Immunocytochemistry (IF-cell)

Species: Rat
Sample: C6 (Rat glioma cell)

Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.
Permeabilization: 0.1% Triton X-100, 15 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 1 hour at room temperature.
Antibody dilution buffer: 1% BSA in PBST.

Primary antibody: HA724555, 1/500, overnight at 4°C.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 45 minutes at room temperature.
Counterstain: Beta tubulin (HA601187, Red), 1/100, overnight at 4℃. The nuclear counterstain was DAPI (Blue).

Application: Immunohistochemistry (IHC-P)

Species: Mouse
Tissue: Testis
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95°C.

Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA724555, 1/100, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Rat
Tissue: Testis
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95°C.

Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA724555, 1/100, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Flow Cytometry (Intra)

Species: Human
Sample: HeLa (Human cervix adenocarcinoma epithelial cell)

Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.
Permeabilization: 0.1% Tween-20, 15 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 15 minutes at room temperature.
Antibody dilution buffer: 1x PBS.

Primary antibody: HA724555 (1/1,000, Red) compared with Rabbit IgG Isotype Control (HA722127, Green), 15 minutes at room temperature.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 15 minutes at room temperature.

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells with Histone H3 (tri methyl K4) (HA724555) / Competitor's antibody / 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.