Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free

10 product images

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  Image from Shan M et al., PLoS One. 2013;8(10):e76408. Fig 1.; doi: 10.1371/journal.pone.0076408. eCollection 2013. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  Formalin-fixed, paraffin-embedded human normal breast, luminal-A DCIS (ductal carcinoma in situ) and triple negative breast cancer tissues stained for CDKN2A/p16INK4a using Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349 in immunohistochemical analysis.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349).

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  Western blot - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  Blocking buffer and concentration: 5% NFDM/TBST
  

  Diluting buffer and concentration: 5% NFDM/TBST

  All lanes: Western blot - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  All lanes: HEK293 (human embryonic kidney) whole cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051)

  Predicted band size: 17 kDa

  Exposure time: 3min

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervix carcinoma tissue labeling CDKN2A/p16INK4a with purified Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, an 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.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349).

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  Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349 (purified) at 1/30 immunoprecipitating CDKN2A/p16INK4a in HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate. For western blotting, a peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/1000).

  Blocking/Dilution buffer: 5% NFDM/TBST.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349).

  All lanes: Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349)

  Predicted band size: 17 kDa

  Observed band size: 17 kDa

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  Flow Cytometry (Intracellular) - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  Overlay histogram showing HEK-293 (Human epithelial cell line from embryonic kidney) cells stained with unpurified Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349 (red line). The cells were fixed with 4% paraformaldehyde (10 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. 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 (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349, 1/100) for 30 minutes at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK-293 cells fixed with 80% methanol (5 minutes)/permeabilized with 0.1% PBS-Tween for 20 minutes used under the same conditions.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labeling CDKN2A/p16INK4a with unpurified Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349 at a dilution of 1/250.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349).

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  Flow Cytometry (Intracellular) - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  Flow Cytometry analysis of HEK-293 (Human epithelial cell line from embryonic kidney) cells labeling CDKN2A/p16INK4a with purified Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349 at 1/270 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabeled control, cells without incubation with primary and secondary antibodies.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349).

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  Western blot - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  This data was developed using the same antibody clone in a different buffer formulation (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349).

  Western blot: Anti-CDKN2A antibody [EPR1473] (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349) staining at 1/4000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349 was shown to bind specifically to CDKN2A. A band was observed at 17 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in CDKN2A knockout cell line. To generate this image, wild-type and CDKN2A knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349) at 1/4000 dilution

  Lane 1: Wild-type HEK-293T cell lysate at 20 µg

  Lane 2: CDKN2A knockout HEK-293T cell lysate at 20 µg

  Lane 3: HeLa cell lysate at 20 µg

  Lane 4: MCF7 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

  Performed under reducing conditions.

  Predicted band size: 17 kDa

  Observed band size: 17 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  Tissue Microarrays stained for "Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal" using "Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. The sections were incubated with Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

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  Flow Cytometry (Intracellular) - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (ab186932)

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349).
  Flow cytometry overlay histogram showing left HeLa positive cells and right negative MCF7 stained with Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal ab108349) (1x 106 in 100μl at 0.04μg/ml (1/52500)) for 30min at 22°C.
  
  The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C
  
  Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.
  
  Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.