Anti-p21 抗体 [EPR362]

• WB

Lab

Western blot - Anti-p21 antibody [EPR362] (AB109520)

Western blot : Anti-CDKN1A antibody [EPR362] (ab109520) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109520 was shown to bind specifically to CDKN1A. A band was observed at 21 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CDKN1A knockout cell line. To generate this image, wild-type and CDKN1A 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-p21 antibody [EPR362] (ab109520) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

CDKN1A knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type MCF7 cell lysate at 20 µg

Lane 4:

CDKN1A knockout MCF7 cell lysate at 20 µg

Lane 5:

Wild-type A549 cell lysate at 20 µg

Lane 6:

CDKN1A knockout A549 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 21 kDa

Observed band size: 21 kDa

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• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-p21 antibody [EPR362] (AB109520)

Intracellular flow cytometric analysis of 90% methanol permeabilized HeLa cells labelling p21 with ab109520 at a dilution of 1/100 (red). Cells were fixed with 4% paraformaldehyde. Goat anti-Rabbit IgG H&L (Alexa Fluor® 488, ab150081) preadsorbed (1/5000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG (ab172730). Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p21 antibody [EPR362] (AB109520)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue labelling p21 with purified ab109520 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p21 antibody [EPR362] (AB109520)

Immunocytochemistry/Immunofluorescence analysis of MCF7 cells labeling p21 with purified ab109520 at 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/1000) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p21 antibody [EPR362] (AB109520)

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma cell line) labeling p21 with ab109520 at 1/500 (2 μg/ml). ab150077, Alexa Fluor^®488 Goat anti-Rabbit at 1/1000 (2 μg/ml) was used as secondary antibody. ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/ml) was used as counterstain AbID. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Nuclei were counter stained blue with DAPI.

Confocal image showing nuclear staining on MCF7 cell line.

• IP

Lab

Immunoprecipitation - Anti-p21 antibody [EPR362] (AB109520)

ab109520 (purified) at 1/50 immunoprecipitating p21 in HEK293 whole cell lysate.

Lane 1 (input) : HEK293 whole cell lysate (10μg)

Lane 2 (+) : ab109520 + HEK293 whole cell lysate (10μg).

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109520 in HEK293 whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1500 dilution.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-p21 antibody [EPR362] (ab109520)

Predicted band size: 18 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-p21 antibody [EPR362] (AB109520)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of HEK-293 whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab109520 (1/2000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique.

ab109520 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected under these conditions. This data was generated as part of a structured antibody stability study supporting Abcam’s transition from cold‑chain to ambient shipping.

All lanes:

Western blot - Anti-p21 antibody [EPR362] (ab109520) at 1/2000 dilution

All lanes:

HEK-293 whole cell lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

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Exposure time: 40s

• WB

Lab

Western blot - Anti-p21 antibody [EPR362] (AB109520)

Different batches of ab109520 were tested on MCF7 (Human breast adenocarcinoma epithelial cell) lysate at 0.2 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 21 kDa.

All lanes:

Western blot - Anti-p21 antibody [EPR362] (ab109520)

Predicted band size: 18 kDa

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• WB

Lab

Western blot - Anti-p21 antibody [EPR362] (AB109520)

False colour image of Western blot : Anti-p21 antibody [EPR362] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109520 was shown to bind specifically to p21. A band was observed at 21 kDa in wild-type HeLa cell lysates with no signal observed at this size in CDKN1A knockout cell line ab255349 (knockout cell lysate ab263812). To generate this image, wild-type and CDKN1A knockout 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-p21 antibody [EPR362] (ab109520) at 1/1000 dilution

Lane 1:

wild-type HeLa Vehicle Control Fluvastatin (20 uM, 24 h) cell lysate at 20 µg

Lane 2:

wild-type HeLa Treated Fluvastatin (50 uM, 24 h) cell lysate at 20 µg

Lane 3:

CDKN1A knockout HeLa Vehicle Control Fluvastatin (20 uM, 24 h) cell lysate at 20 µg

Lanes 3 - 4:

Western blot - Human CDKN1A knockout HeLa cell line (<a href='/products/cell-lines/human-cdkn1a-knockout-hela-cell-line-ab255349'>ab255349</a>)

Lane 4:

CDKN1A knockout HeLa Treated Fluvastatin (50 uM, 24 h) cell lysate at 20 µg

Lane 5:

MCF7 cell lysate at 20 µg

Lane 6:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 18 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-p21 antibody [EPR362] (AB109520)

Lane 1 : Wild-type DLD-1 cell lysate (20 μg)

Lane 2 : Wild-type DLD-1 20 μM 2,3-DCPE for 16hrs treated cell lysate (20 μg)

Lane 3 : p21 knockout DLD-1 cell lysate (20 μg)

Lane 4 : p21 knockout 20 μM 2,3-DCPE for 16hrs DLD-1 cell lysate (20 μg)

Lane 5 : HT1080 cell lysate (20 μg)

Lanes 1 - 5 : Merged signal (red and green). Green - ab109520 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109520 was shown to specifically recognize p21 in wild-type DLD-1 cells treated with 20 μM 2,3-DCPE. No band was observed when p21 knockout samples +/- 2,3-DCPE treatment were used. Wild-type and p21 knockout samples were subjected to SDS-PAGE. ab109520 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-p21 antibody [EPR362] (ab109520)

Predicted band size: 18 kDa

Observed band size: 20 kDa

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• WB

Lab

Western blot - Anti-p21 antibody [EPR362] (AB109520)

Lane 1 : Wild-type DLD-1 cell lysate (20 μg)
Lane 2 : Wild-type DLD-1 20 μM 2,3-DCPE for 16hrs treated cell lysate (20 μg)
Lane 3 : p21 knockout DLD-1 cell lysate (20 μg)
Lane 4 : p21 knockout 20 μM 2,3-DCPE for 16hrs DLD-1 cell lysate (20 μg)
Lane 5 : HT1080 cell lysate (20 μg)

Lanes 1 - 5 : Merged signal (red and green). Green - ab109520 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab109520 and a competitor's top cited rabbit polyclonal antibody.

All lanes:

Western blot - Anti-p21 antibody [EPR362] (ab109520)

Predicted band size: 18 kDa

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• WB

Lab

Western blot - Anti-p21 antibody [EPR362] (AB109520)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-p21 antibody [EPR362] (ab109520) at 1/2000 dilution

Lane 1:

MCF7 cell lysate at 20 µg

Lane 2:

HEK293 cell lysate at 20 µg

Lane 3:

U87-MG cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 18 kDa,24 kDa,57 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-p21 antibody [EPR362] (AB109520)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-p21 antibody [EPR362] (ab109520) at 1/10000 dilution

All lanes:

LnCaP cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 18 kDa

Observed band size: 21 kDa

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