p21 Recombinant Rabbit Monoclonal Antibody [JE00-63]

Western blot analysis of p21 on different lysates with Rabbit anti-p21 antibody (HA722685) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HUVEC cell lysate
Lane 3: MCF7 cell lysate
Lane 4: HCT 116 cell lysate
Lane 5: A549 cell lysate
Lane 6: LNCaP cell lysate
Lane 7: 293T cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 18 kDa
Observed band size: 21 kDa

Exposure time: 3 minutes; ECL: K1801;

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 (HA722685) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of MCF7 cells labeling p21 with Rabbit anti-p21 antibody (HA722685) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-p21 antibody (HA722685) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-p21 antibody (HA722685) at 1/50 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 (HA722685) at 1/50 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 human cervical cancer tissue with Rabbit anti-p21 antibody (HA722685) at 1/50 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 (HA722685) at 1/50 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.

p21 was immunoprecipitated from 0.2 mg MCF7 cell lysate with HA722685 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722685 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: MCF7 cell lysate (input)
Lane 2: HA722685 IP in MCF7 cell lysate
Lane 3: Rabbit IgG instead of HA722685 in MCF7 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 1 minute 31 seconds; ECL: K1801