Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade

• ChIP

Unknown

ChIP - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (AB217876)

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

• IP

Supplier Data

Immunoprecipitation - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (AB217876)

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:

Immunoprecipitation - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (ab217876)

Predicted band size: 94 kDa

Observed band size: 94 kDa

false

• WB

Supplier Data

Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (AB217876)

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:

Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (ab217876) at 1/1000 dilution

Lane 1:

Wild type HAP1 whole cell lysate at 10 µg

Lane 2:

KAT2A / GCN5-knockout HAP1 whole cell lysate whole cell lysate at 10 µg

Lane 3:

HEK-293T (uman epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 10 µg

Lane 4:

MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 10 µg

Predicted band size: 94 kDa

Observed band size: 94 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (AB217876)

False colour image of Western blot : Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab217876 was shown to bind specifically to KAT2A / GCN5. A band was observed at 95 kDa in wild-type U-2 OS cell lysates with no signal observed at this size in kat2a knockout cell line. To generate this image, wild-type and kat2a knockout U-2 OS cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (ab217876) at 1/1000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 20 µg

Lane 2:

GCN5 knockout U-2 OS cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

NIH/3T3 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 94 kDa

Observed band size: 95 kDa

false

• WB

Lab

Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (AB217876)

Western blot : Anti-KAT2A antibody [EPR21146] (ab217876) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab217876 was shown to bind specifically to KAT2A. A band was observed at 95 kDa in wild-type A549 cell lysates with no signal observed at this size in KAT2A knockout cell line. To generate this image, wild-type and KAT2A knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (ab217876) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

KAT2A knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type U-2 OS cell lysate at 20 µg

Lane 4:

GCN5 knockout U-2 OS cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

false

• WB

Lab

Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (AB217876)

Western blot : Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab217876 was shown to bind specifically to KAT2A / GCN5. A band was observed at 94 kDa in wild-type U-2 OS cell lysates with no signal observed at this size in kat2a knockout cell line. To generate this image, wild-type and kat2a knockout U-2 OS cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (ab217876) at 1/1000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 20 µg

Lane 2:

GCN5 knockout U-2 OS cell lysate at 20 µg

Observed band size: 94 kDa

false

• WB

Supplier Data

Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (AB217876)

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.

All lanes:

Western blot - 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 at 10 µg

Lane 2:

Human fetal brain tissue lysate at 10 µg

Secondary

Lane 1:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Lane 2:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 94 kDa

Observed band size: 94 kDa

false

• WB

Supplier Data

Western blot - Anti-KAT2A / GCN5 antibody [EPR21146] - ChIP Grade (AB217876)

Blocking/Dilution buffer : 5% NFDM/TBST

Exposure time : 1 second

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

All lanes:

Western blot - 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:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 94 kDa

Observed band size: 27 kDa

true