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Synthetic peptide corresponding to Human p27 KIP 1 aa 150-250 (C terminal).
A trial size is available to purchase for this antibody.
Mouse: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents
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 ab32034 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-Fr||Use at an assay dependent concentration.|
The rat recommendation is based on the WB results. We do not guarantee IHC-P for rat.
|IHC-FrFl||Use at an assay dependent concentration. PubMed: 23880266|
|WB||1/5000. Detects a band of approximately 27 kDa (predicted molecular weight: 22 kDa).
For unpurified use at 1:1000.
|ICC/IF||1/100 - 1/500.|
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
For unpurified use at 1/20 - 1/40.
For unpurified use at 1:50.
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.
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 secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.
Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling p27 KIP 1 with purified ab32034 at 1:50 dilution (10 µg/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
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.
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 1 in the human cell line MCF-7(human breast carcinoma) by 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.
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 staining p27 KIP 1 in human stomach tissue sections by Immunohistochemistry (frozen sections). Tissue was fixed with acetone and then blocked with 5% serum for 1 hour at 23°C followed by incubation with the primary antibody, at a 1/100 dilution, for 1 hour at 23°C. An undiluted HRP-conjugated goat polyclonal was used as secondary antibody.
Unpurified ab32034 showing positive staining in Colonic adenocarcinoma tissue.
Unpurified ab32034 showing positive staining in Glioma tissue.
Unpurified ab32034 showing positive staining in Stomach adenocarcinoma tissue.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"