Histone H4 (acetyl K12) Recombinant Rabbit Monoclonal Antibody [PSH25-67]

☑ Cell treatment (CT)

Western blot analysis of Histone H4 (acetyl K12) on different lysates with Rabbit anti-Histone H4 (acetyl K12) antibody (HA724554) at 1/50,000 dilution.

Lane 1: HeLa (Human cervical adenocarcinoma cell) cell lysate
Lane 2: HeLa treated with 1μM TSA for 24 hours cell lysate
Lane 3: NIH/3T3 (Mouse fibroblast) cell lysate
Lane 4: NIH/3T3 treated with 400nM TSA for 18 hours cell lysate
Lane 5: C6 (Rat glioma cell) cell lysate
Lane 6: C6 treated with 1μM TSA for 18 hours cell lysate

Lysates/proteins at 10 µg/Lane.
Exposure time: 2 seconds; ECL: K1801

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA724554, 1/50,000 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: 11 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: HA724554, 1/2,000, 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: HA724554, 1/2,000, 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: HA724554, 1/2,000, 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: Human
Tissue: Brain
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: HA724554, 1/500, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Mouse
Tissue: Colon
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: HA724554, 1/1,000, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Rat
Tissue: Colon
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: HA724554, 1/1,000, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Immunoprecipitation (IP)

Histone H4 (acetyl K12) was immunoprecipitated in 0.2 mg HeLa (Human cervix adenocarcinoma epithelial cell) cell lysate with HA724554 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA724554 at 1/5,000 dilution. Anti-Rabbit IgG for IP, AlpSdAbs® VHH(HRP) (025-100-005) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: HA724554 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of HA724554 in HeLa cell lysate

Exposure time: 2 seconds
Blocking: 5% NFDM/TBST, 1 hour at room temperature

Primary dilution: HA724554, 1/5,000 in primary antibody dilution buffer (K1803), 2 hours at room temperature

Predicted band size: 11 kDa
Observed band size: 15 kDa

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells with Histone H4 (acetyl K12) (HA724554) / 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.