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

• Flow Cyt (Intra)

Lab

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 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 (ab108349).

• Flow Cyt (Intra)

Lab

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 (ab108349).

Flow cytometry overlay histogram showing left HeLa positive cells and right negative MCF7 stained with 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 (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.

• Flow Cyt (Intra)

Unknown

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 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 (ab108349, 1/100) for 30 minutes at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 minutes at 22°C. 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 (ab108349).

• IHC-P

PubMed

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 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 (ab108349).

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/

• IHC-P

Lab

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 ab108349 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. 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 (ab108349).

• IHC-P

Unknown

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 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 (ab108349).

• IP

Lab

Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (AB186932)

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 (ab108349).

All lanes:

Immunoprecipitation - Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal (<a href='/en-us/products/primary-antibodies/cdkn2a-p16ink4a-antibody-epr1473-c-terminal-ab108349'>ab108349</a>)

Predicted band size: 17 kDa

Observed band size: 17 kDa

false

• WB

Lab

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 (ab108349).

Western blot : Anti-CDKN2A antibody [EPR1473] (ab108349) staining at 1/4000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, 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 (<a href='/en-us/products/primary-antibodies/cdkn2a-p16ink4a-antibody-epr1473-c-terminal-ab108349'>ab108349</a>) 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

Predicted band size: 17 kDa

Observed band size: 17 kDa

false

• WB

Lab

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>)

Predicted band size: 17 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR1473] - BSA and Azide free (AB186932)

This data was developed using ab108349, the same antibody clone in a different buffer formulation.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/1000000 dilution.

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

All lanes:

Western blot - Anti-CDKN2A/p16INK4a antibody [EPR1473] - C-terminal (<a href='/en-us/products/primary-antibodies/cdkn2a-p16ink4a-antibody-epr1473-c-terminal-ab108349'>ab108349</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T (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:

HUVEC (human umbilical vein endothelial 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 1/20000 dilution

Observed band size: 16 kDa,36 kDa

false

Exposure time: 120s

• IHC-P

Lab

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 "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 ab108349 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.