Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12]

• IP

Supplier Data

Immunoprecipitation - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (AB306579)

KDM5B / PLU1 / Jarid1B was immunoprecipitated from 0.35 mg Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate 10 ug with ab306579 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab306579 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Wild-type HAP1 whole cell lysate 10 ug
Lane 2 : ab306579 IP in HAP1 whole cell lysate 10 ug
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab306579 in HAP1 cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 180 seconds.

All lanes:

Immunoprecipitation - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (ab306579) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate 10 µg

Lane 2:

HAP1 whole cell lysate 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 180 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (AB306579)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS. The bands beneath the target band (180 kDa) are likely to be degradation products.  Performed under reducing conditions. False colour image of Western blot : Anti-KDM5B / PLU1 / Jarid1B antibody (ab306579) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 loading control staining at 1/20000 dilution, shown in red. In Western blot, ab306579 was shown to bind specifically to KDM5B / PLU1 / Jarid1B. A band was observed at 180 kDa in wild-type HAP1 cell lysates whereas no signal observed at this size in KDM5B / PLU1 / Jarid1B knockout cell line. To generate this image, wild-type and KDM5B / PLU1 / Jarid1B knockout HAP1 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (ab306579) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate 20 µg

Lane 2:

KDM5B / PLU1 / Jarid1B knockout HAP1 whole cell lysate 20 µg

Lane 3:

K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 20 µg

Secondary

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/ko/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Observed band size: 180 kDa

false

• WB

Supplier Data

Western blot - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (AB306579)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. The bands beneath the target band (180 kDa) are likely to be degradation products as this sample was frozen before the Western Blot was run. Exposure time : 37 seconds.

All lanes:

Western blot - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (ab306579) at 1/1000 dilution

All lanes:

Human testis tissue lysate 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Observed band size: 180 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (AB306579)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. This antibody does not cross-react with human KDM5A and KDM5C. Exposure time : 3 seconds.

All lanes:

Western blot - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (ab306579) at 1/1000 dilution

Lane 1:

Human KDM5B recombinant protein 10 ng

Lane 2:

Human KDM5A recombinant protein 10 ng

Lane 3:

Human KDM5C recombinant protein 10 ng

Secondary

All lanes:

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

Observed band size: 180 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (AB306579)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. In lanes 1 and 2, the lysates were stored at -80degC prior to Western Blotting. The bands beneath the target band (180 kDa) are likely to be degradation products. In lanes 3 and 4, the lysates were freshly made and used for Western Blotting immediately to minimize protein degradation. The Lane 4 was developed using a high sensitivity ECL substrate. Exposure time : Lanes 1 and 2 : 15 seconds, Lanes 3 and 4 : 180 seconds.

All lanes:

Western blot - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (ab306579) at 1/1000 dilution

Lane 1:

K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 20 μg

Lane 2:

HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate 20 μg

Lane 3:

HAP1 whole cell lysate 20 μg

Lane 4:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate 20 μg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Observed band size: 180 kDa

true

Exposure time: 15s

• WB

Lab

Western blot - Anti-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (AB306579)

Western blot : Anti-KDM5B antibody [EPR25242-12] (ab306579) 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, ab306579 was shown to bind specifically to KDM5B. A band was observed at 171 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in KDM5B knockout cell line. To generate this image, wild-type and KDM5B knockout HCT 116 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-KDM5B / PLU1 / Jarid1B antibody [EPR25242-12] (ab306579) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human KDM5B knockout HCT116 cell line (<a href='/ko/products/cell-lines/human-kdm5b-knockout-hct116-cell-line-ab287592'>ab287592</a>)

Lane 2:

KDM5B knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

KDM5B knockout HAP1 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 171 kDa

false