Recombinant Anti-p27 KIP 1 antibody [Y236] (ab32034)

False colour image of Western blot: Anti-p27 KIP 1 antibody [Y236] staining at 1/5000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32034 was shown to bind specifically to p27 KIP 1. A band was observed at 28 kDa in wild-type RAW 264.7 cell lysates with no signal observed at this size in CDKN1B knockout cell line ab281619 (knockout cell lysate ab282970). To generate this image, wild-type and CDKN1B knockout RAW 264.7 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 HAP1 whole cell lysate (20 µg)
Lane 2: CDKN1B knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: MCF7 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab32034 observed at 30 kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab32034 was shown to specifically react with CDKN1B in wild-type HAP1 cells. No band was observed when CDKN1B knockout samples were used. Wild-type and CDKN1B knockout samples were subjected to SDS-PAGE.  ab32034 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 and 1/10000 respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cervical carcinoma tissue sections labeling p27 KIP 1 with Purified ab32034 at 1:50 dilution (10.4 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

ab32034 (purified) at 1/30 dilution (20 µg/mL) immunoprecipitating p27 KIP 1 in C6 whole cell lysate.
Lane 1 (input): C6(Rat glial tumor glial cell) whole cell lysate 10 µg
Lane 2 (+): ab32034 & C6 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32034 in C6 whole cell lysate
For western blotting, ab32034 at 1/500 dilution (1.044 µg/mL) and VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM /TBST .

Intracellular Flow Cytometry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labelling p27 KIP 1 with ab32034 at 1/500 dilution (5.22 µg/ml) (Red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlabeled control - Unlabelled cells (Blue).

Overlay histogram showing HAP1 wildtype (green line) and HAP1-CDKN1B knockout cells (red line) stained with ab32034. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab32034, 1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-CDKN1B knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. This antibody can also be used in HAP1 cells fixed with 4%PFA (10 min), permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions. 

ab32034 (purified) at 1/20 dilution (2μg) immunoprecipitating p27 KIP 1 in MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate.

Lane 1 (input): MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+): ab32034 & MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32034 in MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling p27 KIP 1 with purified ab32034 at 1/50 dilution (10µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

Blocking and diluting buffer: 5% NFDM/TBST

Blocking and diluting buffer: 5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded human breast carcinoma. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling p27 KIP 1 (green) with purified ab32034 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).

Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

Unpurified ab32034 staining p27 KIP 1in the human cell line MCF-7 (human breast carcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody. 

Isoytype control: Rabbit monoclonal IgG (Black) 

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue) 

Unpurified ab32034 showing positive staining in Ovarian carcinoma tissue. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Unpurified ab32034 staining p27 KIP1 in MCF7 cells treated with NS 398 (ab120295), by ICC/IF. Increase in p27 KIP1 expression correlates with increased concentration of NS 398, as described in literature.
The cells were incubated at 37°C for 6h in media containing different concentrations of ab120295 (NS 398) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab32034 (1/100 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody.

Unpurified ab32034 showing positive staining in Colonic adenocarcinoma tissue. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Unpurified ab32034 showing positive staining in Glioma tissue. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Unpurified ab32034 showing positive staining in Stomach adenocarcinoma tissue. Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).