All lanes : Anti-HDAC2 antibody [EPR5001] - ChIP Grade (ab124974) at 1/10000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : HDAC2 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 55 kDa
Observed band size: 55 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab124974 observed at 55 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab124974 was shown to react with HDAC2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266589 (knockout cell lysate ab256938) was used. Wild-type HEK-293T and HDAC2 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab124974 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab124974 staining HDAC2 in wild-type HAP1 cells (top panel) and HDAC2 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab124974 at 0.5μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

All lanes : Anti-HDAC2 antibody [EPR5001] - ChIP Grade (ab124974) at 1/10000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : HDAC2 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 55 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Lanes 1-2: Merged signal (red and green). Green - ab124974 observed at 60 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab124974 Anti-HDAC2 antibody [EPR5001] was shown to specifically react with HDAC2 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266590 (knockout cell lysate ab256939) was used. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE. ab124974 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-HDAC2 antibody [EPR5001] - ChIP Grade (ab124974) at 1/10000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : HDAC2 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 55 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Lanes 1-2: Merged signal (red and green). Green - ab124974 observed at 60 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab124974 Anti-HDAC2 antibody [EPR5001] was shown to specifically react with HDAC2 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266588 (knockout cell lysate ab256937) was used. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE. ab124974 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 Dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

 

All lanes : Anti-HDAC2 antibody [EPR5001] - ChIP Grade (ab124974) at 1/10000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : HDAC2 knockout HAP1 whole cell lysate
Lane 3 : A431 whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 55 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab124974 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab124974 was shown to recognize HDAC2 when HDAC2 knockout samples were used, along with additional cross-reactive bands. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE.  ab124974 and ab8245 (Mouse anti GAPDH loading control) were but diluted at 1/10000 and incubated overnight at 4°C. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-HDAC2 antibody [EPR5001] - ChIP Grade (ab124974) at 1/10000 dilution

Lane 1 : HeLa cell lysate
Lane 2 : SH-SY5Y cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 55 kDa

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 10µg of ab124974 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). 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.