ADNP Recombinant Rabbit Monoclonal Antibody [PSH24-23]

☑ Knockout (KO)

Western blot analysis of ADNP on different lysates with Rabbit anti-ADNP antibody (HA724430) at 1/5,000 dilution.

Lane 1: 293T-ADNP-WT cell lysate
Lane 2: 293T-ADNP-KO cell lysate
Lane 3: HeLa (Human cervical adenocarcinoma cell) cell lysate
Lane 4: NIH/3T3 (Mouse fibroblast) cell lysate
Lane 5: C2C12 (Mouse myoblast) cell lysate
Lane 6: PC-12 (Rat pheochromocytoma cell (undifferentiated)) cell lysate
Lane 7: C6 (Rat glioma cell) cell lysate

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

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA724430, 1/2,000 in 5% NFDM/TBST, 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: 124 kDa
Observed band size: 140 kDa

Application: Immunofluorescence (IHC-Fr)

Species: Mouse
Tissue: Brain
Sample: Frozen section

Antigen retrieval: Not required

Wash buffer: 1× TBST
Blocking: 10% normal goat serum + 1% Triton X-100 + 0.3 M Glycine in TBST, 30 minutes at room temperature.

Primary antibody: HA724430, 1/500, overnight at 4°C.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 1.5 hours at room temperature.

Application: Immunofluorescence (IHC-Fr)

Species: Rat
Tissue: Brain
Sample: Frozen section

Antigen retrieval: Not required

Wash buffer: 1× TBST
Blocking: 10% normal goat serum + 1% Triton X-100 + 0.3 M Glycine in TBST, 30 minutes at room temperature.

Primary antibody: HA724430, 1/500, overnight at 4°C.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 1.5 hours at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: Colon cancer
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: HA724430, 1/2,000, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Mouse
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: HA724430, 1/2,000, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Rat
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: HA724430, 1/2,000, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunocytochemistry (IF-cell)

Species: Human
Sample: HeLa (Human cervix adenocarcinoma epithelial 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: HA724430, 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: Neuro-2a (Mouse brain neuroblastoma 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: HA724430, 1/100, 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: HA724430, 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).

Immunoprecipitation (IP)

ADNP was immunoprecipitated in 0.2 mg Neuro-2a (Mouse brain neuroblastoma cell) cell lysate with HA724430 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA724430 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: Neuro-2a cell lysate (input)
Lane 2: HA724430 IP in Neuro-2a cell lysate
Lane 3: Rabbit IgG instead of HA724430 in Neuro-2a cell lysate

Exposure time: 59 seconds
Blocking: 5% NFDM/TBST, 1 hour at room temperature

Primary dilution: HA724430, 1/1,000 in primary antibody dilution buffer (K1803), 2 hours at room temperature

Predicted band size: 124 kDa
Observed band size: 140 kDa

Chromatin immunoprecipitations were performed with cross-linked chromatin from Neuro-2a cells with ADNP (HA724430) / 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.