GATA2 was immunoprecipitated from 0.35 mg K-562 (Human chronic myelogenous leukemia lymphoblast) cell lysate 10 µg with ab109241 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab109241 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: K-562 (Human chronic myelogenous leukemia lymphoblast) cell lysate 10 µg

Lane 2: ab109241 IP in K-562 cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab109241 in K562 cell lysate

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

Exposure time: 8 seconds

All lanes : Anti-GATA2 antibody [EPR2822(2)] (ab109241) at 1 µg/ml

Lane 1 : Wild-type HEK-293 whole cell lysate
Lane 2 : GATA2 knockout HEK-293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 51 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab109241 observed at 50 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109241 was shown to recognize GATA2 in wild-type HEK-293 cells as signal was lost at the expected MW in GATA2 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GATA2 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab109241 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml 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 1/20000 dilution for 1 hour at room temperature before imaging.

Purified ab109241 at 1/30 dilution (2 µg) immunoprecipitating GATA2 in K-562 whole cell lysate.
Lane 1 (input): K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 µg
Lane 2 (+): ab109241 + K-562 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109241 in K-562 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 51 kDa

All lanes : Anti-GATA2 antibody [EPR2822(2)] (ab109241) at 1/1000 dilution (purified)

Lane 1 : bEnd.3 (Mouse brain endothelioma) whole cell lysate
Lane 2 : Mouse placenta lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 51 kDa
Observed band size: 51 kDa

All lanes : Anti-GATA2 antibody [EPR2822(2)] (ab109241) at 1/1000 dilution

Lane 1 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate prepared using 1% SDS hot lysis method
Lane 2 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate prepared using RIPA lysis method
Lane 3 : U-937 (Human histiocytic lymphoma monocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 51 kDa


Exposure time: 3 minutes

Blocking and diluting buffer: 5% NFDM/TBST.

The different result in K-562 is due to the lysates preparation method.

For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

The expression profile observed in U-937 is consistent with the literature (PMID: 19212333).
Negative control: U-937 (PMID: 19212333)