Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: p73 knockout HAP1 whole cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab215038 observed at 75 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab215038 was shown to recognize p73 in wild-type HAP1 cells as signal was lost at the expected MW in p73 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and p73 knockout samples were subjected to SDS-PAGE. Ab215038 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/50 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-p73 antibody [EPR19884] - ChIP Grade (ab215038) at 1/1000 dilution

Lane 1 : HT-1376 (Human urinary bladder carcinoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : 293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 4 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 70 kDa
Observed band size: 70,80 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1: 3 minutes; Lane 2-4: 30 seconds.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 11101847).

Immunohistochemical analysis of paraffin-embedded human skin tissue labeling p73 with ab215038 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on basal and parabasal layers of squamous epithelium of human skin is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling p73 with ab215038 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on basal and parabasal layers of squamous epithelium of human tonsil is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling p73 with ab215038 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human bladder cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human lung squamous carcinoma tissue labeling p73 with ab215038 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human lung squamous carcinoma is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

p73 was immunoprecipitated from 0.35 mg of HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate with ab215038 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab215038 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1,000 dilution

Lane 1: HEK-293 whole cell lysate 10 μg (Input).

Lane 2: ab215038 IP in HEK-293 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab215038 in HEK-293 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.

Chromatin was prepared from HCT 116 (Human colorectal carcinoma cell line) cells treated with 1mM Hydroxyurea for 16h and non-treated according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab215038 (blue), and 20µl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow).  The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).