Recombinant Anti-p21 antibody [EPR3993] (ab109199)

False colour image of Western blot: Anti-p21 antibody [EPR3993] 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, ab109199 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 CDKN2A knockout cell line ab255349 (knockout cell lysate ab263812). To generate this image, wild-type and CDKN2A 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 - ab109199 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109199 was shown to recognize p21 in WT DLD-1 cells with 2,3-DCPE treatment along with additional cross-reactive bands. When p21 knockout DLD-1 cells +/- 2,3-DCPE treatment were used, no band was observed. Wild-type and p21 knockout samples were subjected to SDS-PAGE. ab109199 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.

Lanes 1- 2: Merged signal (red and green). Green - ab109199 observed at 20 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109199 was shown to react with p21 in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266860 (knockout cell lysate ab256870) was used. Wild-Type HCT116 and CDKN1A knockout HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109199 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

Blocking and diluting buffer: 5% NDFM/TBST.

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 - ab109199 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab109119 and a competitor's top cited rabbit polyclonal antibody.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.