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Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human p21 aa 1-100. The exact sequence is proprietary.
A trial size is available to purchase for this antibody.
Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
This product is a recombinant rabbit monoclonal antibody.
Our Abpromise guarantee covers the use of ab109520 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
For unpurified use at 1/50 - 1/100.
|WB||1/1000 - 1/10000. Predicted molecular weight: 21 kDa.|
|IP||1/10 - 1/100.|
|IHC-P||1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).
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 when p21 knockout samples 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.
Immunocytochemistry/Immunofluorescence analysis of MCF7 cells labelling 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).
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.
Blocking and dilution buffer: 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 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22ºC. 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.
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, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/1500).
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 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.
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.