Anti-KDM5C / Jarid1C / SMCX抗体

• ICC/IF

AbReview47768****

Immunocytochemistry/ Immunofluorescence - Anti-KDM5C / Jarid1C / SMCX antibody (AB34718)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-KDM5C / Jarid1C / SMCX antibody (AB34718)

ab34718 staining KDM5C / Jarid1C / SMCX in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab34718 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Project

Western blot - Anti-KDM5C / Jarid1C / SMCX antibody (AB34718)

All lanes:

Western blot - Anti-KDM5C / Jarid1C / SMCX antibody (ab34718) at 1/250 dilution

All lanes:

Western blot - HEK-293 whole cell lysate (<a href='/products/cell-lysates/hek-293-whole-cell-lysate-ab7902'>ab7902</a>) at 20 µg

Secondary

All lanes:

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

Predicted band size: 175 kDa

Observed band size: 176 kDa

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• WB

Ap

Western blot - Anti-KDM5C / Jarid1C / SMCX antibody (AB34718)

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.

All lanes:

Western blot - Anti-KDM5C / Jarid1C / SMCX antibody (ab34718) at 1 µg/mL

Lane 1:

HEK293 (Human) Whole Cell Lysate at 20 µg

Lane 2:

Y79 (Human retinoblastoma cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 175 kDa

Observed band size: 180 kDa,45 kDa,58 kDa

true

Exposure time: 20min

• WB

Lab

Western blot - Anti-KDM5C / Jarid1C / SMCX antibody (AB34718)

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.

All lanes:

Western blot - Anti-KDM5C / Jarid1C / SMCX antibody (ab34718)

Predicted band size: 175 kDa

false

• WB

Lab

Western blot - Anti-KDM5C / Jarid1C / SMCX antibody (AB34718)

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:

Western blot - Anti-KDM5C / Jarid1C / SMCX antibody (ab34718) at 1/250 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

KDM5C knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human KDM5C (Jarid1C / SMCX) knockout HEK-293T cell line (<a href='/products/cell-lines/human-kdm5c-jarid1c-smcx-knockout-hek-293t-cell-line-ab266251'>ab266251</a>)

Predicted band size: 175 kDa

Observed band size: 175 kDa

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