Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: KDM5C knockout HAP1 whole cell lysate (20 µg)
Lane 3: HEK293 whole cell lysate (20 µg)
Lane 4: U2OS whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab34718 observed at 175 kDa. Red - loading control, ab18058, observed at 120 kDa.

ab34718 was shown to specifically recognize KDM5C in wild-type HAP1 cells as signal was lost at the expected MW in KDM5C knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and KDM5C knockout samples were subjected to SDS-PAGE. Ab34718 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/250 dilution and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.

PFA-fixed, 0.5% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained for KDM5C / Jarid1C / SMCX (green) using ab34718 at 1/200 dilution in ICC/IF. Counter-stained with DAPI in order to highlight the nucleus (red). Please refer to abreview for further experimental details.

See Abreview

ab34718 stained in Hela cells. Cells were fixed with 100% methanol (5min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab34718 at 5µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 µg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

All lanes : Anti-KDM5C / Jarid1C / SMCX antibody (ab34718) at 1/250 dilution

Lane 1 : HEK-293T cell lysate
Lane 2 : KDM5C knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 176 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab34718 observed at 175 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab34718 was shown to react with KDM5C / Jarid1C / SMCX in wild-type HEK-293T cells in western blot with loss of signal observed in KDM5C knockout cell line ab266251 (KDM5C knockout cell lysate ab257494). Wild-type and KDM5C knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab34718 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 250 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-KDM5C / Jarid1C / SMCX antibody (ab34718) at 1 µg/ml

Lane 1 : HEK293 (Human) Whole Cell Lysate
Lane 2 : Y79 (Human retinoblastoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 176 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?
Additional bands at: 45 kDa, 58 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab34718 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Anti-KDM5C / Jarid1C / SMCX antibody (ab34718) at 1/250 dilution + HEK-293 whole cell lysate (ab7902) at 20 µg

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 176 kDa
Observed band size: 176 kDa