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 - ab202474 observed at 75 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab202474 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. Ab202474 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-p73 antibody [EPR19560] - ChIP Grade (ab202474) at 1/1000 dilution

Lane 1 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : MDA-MB-231 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 3 : HT-29 (Human colorectal adenocarcinoma cell line) whole cell lysate
Lane 4 : HEK-293T (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 5 : HT-1376 (Human urinary bladder carcinoma cell line) whole cell lysate
Lane 6 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Lanes 5-6 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 69 kDa
Observed band size: 70,80 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/3/4/5: 1 minute; Lane 2/6: 3 minutes.

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

Anti-p73 antibody [EPR19560] - ChIP Grade (ab202474) at 1/1000 dilution + U937 (Human histiocytic lymphoma cell line) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 69 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

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

p73 was immunoprecipitated from 0.35mg of HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate with ab202474 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab202474 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HepG2 whole cell lysate 10µg (Input).

Lane 2: ab202474 IP in HepG2 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab202474 in HepG2 whole cell lysate.

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

Exposure time: 3 minutes.

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 ab202474 (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).