重组Anti-GATA2抗体[EPR2822(2)] (ab109241)

Chromatin was prepared from K-562 (Human chronic myelogenous leukemia lymphoblast) cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with 1.5 mM EGS for 30mins and then 1% formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab109241 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR ( Sybr green approach).
Primers and probes are from paper PMID :22036566.

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

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

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)