Keap1 Recombinant Rabbit Monoclonal Antibody [PSH0-90]

Western blot analysis of Keap1 on different lysates with Rabbit anti-Keap1 antibody (HA721525) at 1/1,000 dilution.

Lane 1: HepG2 cell lysate
Lane 2: HeLa cell lysate
Lane 3: MCF7 cell lysate
Lane 4: A431 cell lysate
Lane 5: Jurkat cell lysate
Lane 6: HEK-293 cell lysate
Lane 7: Daudi cell lysate
Lane 8: A549 cell lysate
Lane 9: NIH/3T3 cell lysate
Lane 10: PC-12 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 70 kDa
Observed band size: 55-70 kDa

Exposure time: 2 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721525) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-Keap1 antibody (HA721525) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA721525) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-Keap1 antibody (HA721525) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA721525) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Flow cytometric analysis of PC-12 cells labeling Keap1.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA721525, 1ug/ml) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

☑ Knockdown (KD)

Western blot analysis of Keap1 on different lysates with Rabbit anti-Keap1 antibody (HA721525) at 1/1,000 dilution.

Lane 1: HepG2-si NT cell lysate
Lane 2: HepG2-si Keap1 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 70 kDa
Observed band size: 55-70 kDa

Exposure time: 1 minute 55 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721525) at 1/1,000 dilution was used in 5% BSA at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Keap1 was immunoprecipitated from 0.2 mg HeLa cell lysate with HA721525 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA721525 at 1/1,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: HA721525 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of HA721525 in HeLa cell lysate

Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 12 seconds; ECL: K1801