Recombinant 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.

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.

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.

Confocal image showing nuclear staining on MCF7 cell line. 

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.

Overlay histogram showing HeLa cells stained with unpurified ab109520 (red line). The cells were fixed with 80% methanol (5 min) then permeabilized with 0.1% PBS-Tween 20 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab109520, 1/100) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730, 1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. 

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).

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.

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human papillary carcinoma of the thyroid gland tissue labelling p21 with unpurified ab109520 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling p21 with unpurified ab109520 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.