Anti-CDKN2A/p16INK4a antibody [EPR24167-43]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

Immunohistochemical analysis of paraffin-embedded Human cervical cancer tissue labelling CDKN2A/p16INK4a with ab270058 at 1/500 (0.936 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in human cervical cancer (PMID : 9736404). The section was incubated with ab270058 for 30 minutes at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 minutes.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling CDKN2A/p16INK4a with ab270058 at 1/50 (9.36 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear and cytoplasmic staining in HeLa cell line. Negative control : MCF7 (PMID : 10914724) is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

Immunohistochemical analysis of paraffin-embedded Human endometrial cancer tissue labelling CDKN2A/p16INK4a with ab270058 at 1/500 (0.936 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in human endometrial cancer. The section was incubated with ab270058 for 30 minutes at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 minutes.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized MCF7 ( Human breast adenocarcinoma epithelial cell, Left)/ HeLa (Human cervix adenocarcinoma epithelial cell, Right) cells labelling CDKN2A/p16INK4a with ab270058 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Negative control : MCF7 (PMID : 10914724).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

Immunohistochemical analysis of paraffin-embedded Human cervix tissue labelling CDKN2A/p16INK4a with ab270058 at 1/500 (0.936 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : No staining in human cervix (PMID : 9736404). The section was incubated with ab270058 for 30 minutes at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 minutes.

• IP

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Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

CDKN2A/p16INK4a was immunoprecipitated from 0.35 mg HEK-293 (human embryonic kidney epithelial cell), whole cell lysate 10 ug with ab270058 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270058 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HEK-293 (human embryonic kidney epithelial cell), whole cell lysate 10 ug

Lane 2 : ab270058 IP in HEK-293 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab270058 in HEK-293 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 32 seconds

All lanes:

Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (ab270058)

Predicted band size: 17 kDa

Observed band size: 16 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

CDKN2A/p16INK4a was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate 10 ug with ab270058 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270058 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate 10 ug

Lane 2 : ab270058 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab270058 in HeLa whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 32 seconds

All lanes:

Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (ab270058)

Predicted band size: 17 kDa

Observed band size: 16 kDa

false

• WB

Lab

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

Western blot : Anti-CDKN2A antibody [EPR24167-43] (ab270058) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab270058 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 [EPR24167-43] (ab270058) at 1/2000 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

Predicted band size: 17 kDa

Observed band size: 17 kDa

false

• WB

Lab

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Negative control : MCF7, MDA-MB-231 (PMID : 10914724)

The MW observed is consistent with the literature. (PMID : 18053084)

This blot was developed using a higher sensitivity ECL substrate.

Exposure time : 70 seconds.

All lanes:

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (ab270058) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

HEK-293 (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 3:

Saos-2 (human osteosarcoma epithelial), whole cell lysate at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 5:

MDA-MB-231 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 17 kDa

Observed band size: 16 kDa

false

• WB

Lab

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

This blot was produced using 14% SDS-PAGE containing 15 μg of HeLa whole cell lysate per lane at 150V for 50 minutes before being transferred onto a 0.45 μm PVDF membrane at 72V for 50 minutes. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab270058 (1/1000) 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 1/100,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab270058 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.

All lanes:

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (ab270058) at 1/1000 dilution

All lanes:

HeLa whole cell lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

Western blot : Anti-CDKN2A antibody [EPR24167-43] (ab270058) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab270058 was shown to bind specifically to CDKN2A. 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-CDKN2A/p16INK4a antibody [EPR24167-43] (ab270058) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 20 µg

Lane 2:

HEK-293 cell lysate at 20 µg

Lane 3:

Saos-2 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Observed band size: 17 kDa

true

• WB

Lab

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (AB270058)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

This antibody specifically recognizes CDKN2A recombinant protein, but not CDKN2B recombinant protein.

Exposure time : 3.25 seconds.

All lanes:

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR24167-43] (ab270058) at 1/1000 dilution

Lane 1:

His-tagged human CDKN2A recombinant protein at 0.01 µg

Lane 2:

GST-tagged human CDKN2B recombinant protein at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 17 kDa

Observed band size: 16 kDa

false