All lanes : Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (ab217876) at 1/1000 dilution

Lane 1 : His-tagged human KAT2A / GCN5 recombinant protein (aa86-336), 10 ng
Lane 2 : His-tagged human KAT2B recombinant protein (aa74-326), 10 ng

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

Developed using the ECL technique.

Predicted band size: 94 kDa
Observed band size: 27 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST

Exposure time: 1 second

The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.

Chromatin was prepared from A549 treated with thapsigargin (1µM 12 hours) cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab217876 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

KAT2A / GCN5 was immunoprecipitated from 0.35mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab217876 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab217876 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10ug (Input). 

Lane 2: ab217876 IP in HeLa whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab217876 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.

All lanes : Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (ab217876) at 1/1000 dilution

Lane 1 : Wild type HAP1 whole cell lysate
Lane 2 : KAT2A / GCN5-knockout HAP1 whole cell lysate whole cell lysate
Lane 3 : HEK-293T (uman epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 4 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 94 kDa
Observed band size: 94 kDa


Exposure time: 3 minutes

ab217876 was shown to specifically react with KAT2A / GCN5 in wild-type HAP1 cells as signal was lost in KAT2A / GCN5 knockout cells. Wild-type and KAT2A / GCN5 knockout samples were subjected to SDS-PAGE. Ab217876 and ab181602 (Human anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/200000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrumentusing the ECL technique.

All lanes : Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (ab217876) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : Human fetal brain tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lane 1 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 2 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 94 kDa
Observed band size: 94 kDa

Blocking/Dilution buffer: 5% NFDM/TBST

Exposure time: Lane 1: 15 seconds; Lane 2: 3 minutes

The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.