Overview

  • Product name
    Anti-p27 KIP 1 antibody [Y236] - BSA and Azide free
    See all p27 KIP 1 primary antibodies
  • Description
    Rabbit monoclonal [Y236] to p27 KIP 1 - BSA and Azide free
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-Fr, IHC-P, IHC-FrFl, WB, ICC/IF, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Rat, Human
  • Immunogen

    Synthetic peptide within Human p27 KIP 1 aa 150-250 (C terminal). The exact sequence is proprietary.

  • Positive control
    • MCF-7 cell lysate, PC-12 cells, human breast carcinoma
  • General notes

    The formulation and the concentration of this product is compatible for metal-conjugation for mass cytometry (CyTOF®).

    ab206927 is a PBS-only buffer version of ab32034, containing no BSA or sodium azide, ideal for antibody labeling. Please refer to ab32034 for information on protocols, dilutions, and image data.

    Mouse: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab206927 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-Fr Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
IHC-FrFl Use at an assay dependent concentration. PubMed: 23880266
WB Use at an assay dependent concentration. Detects a band of approximately 27 kDa (predicted molecular weight: 22 kDa).
ICC/IF Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

 

IP Use at an assay dependent concentration.

Target

  • Function
    Important regulator of cell cycle progression. Involved in G1 arrest. Potent inhibitor of cyclin E- and cyclin A-CDK2 complexes. Forms a complex with cyclin type D-CDK4 complexes and is involved in the assembly, stability, and modulation of CCND1-CDK4 complex activation. Acts either as an inhibitor or an activator of cyclin type D-CDK4 complexes depending on its phosphorylation state and/or stoichometry.
  • Tissue specificity
    Expressed in all tissues tested. Highest levels in skeletal muscle, lowest in liver and kidney.
  • Involvement in disease
    Defects in CDKN1B are the cause of multiple endocrine neoplasia type 4 (MEN4) [MIM:610755]. Multiple endocrine neoplasia (MEN) syndromes are inherited cancer syndromes of the thyroid. MEN4 is a MEN-like syndrome with a phenotypic overlap of both MEN1 and MEN2.
  • Sequence similarities
    Belongs to the CDI family.
  • Domain
    A peptide sequence containing only AA 28-79 retains substantial Kip1 cyclin A/CDK2 inhibitory activity.
  • Post-translational
    modifications
    Phosphorylated; phosphorylation occurs on serine, threonine and tyrosine residues. Phosphorylation on Ser-10 is the major site of phosphorylation in resting cells, takes place at the G(0)-G(1) phase and leads to protein stability. Phosphorylation on other sites is greatly enhanced by mitogens, growth factors, cMYC and in certain cancer cell lines. The phosphorylated form found in the cytoplasm is inactivate. Phosphorylation on Thr-198 is required for interaction with 14-3-3 proteins. Phosphorylation on Thr-187, by CDK2 leads to protein ubiquitination and proteasomal degradation. Tyrosine phosphorylation promotes this process. Phosphorylation by PKB/AKT1 can be suppressed by LY294002, an inhibitor of the catalytic subunit of PI3K. Phosphorylation on Tyr-88 and Tyr-89 has no effect on binding CDK2, but is required for binding CDK4. Dephosphorylated on tyrosine residues by G-CSF.
    Ubiquitinated; in the cytoplasm by the KPC complex (composed of RNF123/KPC1 and UBAC1/KPC2) and, in the nucleus, by SCF(SKP2). The latter requires prior phosphorylation on Thr-187. Ubiquitinated; by a TRIM21-containing SCF(SKP2)-like complex; leads to its degradation.
    Subject to degradation in the lysosome. Interaction with SNX6 promotes lysosomal degradation.
  • Cellular localization
    Nucleus. Cytoplasm. Endosome. Nuclear and cytoplasmic in quiescent cells. AKT-or RSK-mediated phosphorylation on Thr-198, binds 14-3-3, translocates to the cytoplasm and promotes cell cycle progression. Mitogen-activated UHMK1 phosphorylation on Ser-10 also results in translocation to the cytoplasm and cell cycle progression. Phosphorylation on Ser-10 facilitates nuclear export. Translocates to the nucleus on phosphorylation of Tyr-88 and Tyr-89. Colocalizes at the endosome with SNX6 and this leads to lysosomal degradation.
  • Information by UniProt
  • Database links
  • Alternative names
    • AA408329 antibody
    • AI843786 antibody
    • Cdki1b antibody
    • CDKN 1B antibody
    • CDKN 4 antibody
    • CDKN1B antibody
    • CDKN4 antibody
    • CDN1B_HUMAN antibody
    • Cyclin Dependent Kinase Inhibitor 1B antibody
    • Cyclin dependent kinase inhibitor p27 antibody
    • Cyclin-dependent kinase inhibitor 1B (p27, Kip1) antibody
    • Cyclin-dependent kinase inhibitor 1B antibody
    • Cyclin-dependent kinase inhibitor p27 antibody
    • Cyclin-dependent kinase inhibitor p27 Kip1 antibody
    • KIP 1 antibody
    • KIP1 antibody
    • MEN1B antibody
    • MEN4 antibody
    • OTTHUMP00000195098 antibody
    • OTTHUMP00000195099 antibody
    • p27 antibody
    • p27 Kip1 antibody
    • P27-like cyclin-dependent kinase inhibitor antibody
    • p27Kip1 antibody
    see all

Images

  • This WB data was generated using the same anti-p27 KIP 1 antibody clone, Y236, in a different buffer formulation (cat# ab32034).

    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.

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • 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).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • 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)

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • Immunohistochemical analysis of paraffin-embedded human breast carcinoma

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • Unpurified ab32034 showing positive staining in Colonic adenocarcinoma tissue.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • Unpurified ab32034 showing positive staining in Glioma tissue.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • Unpurified ab32034 showing positive staining in Ovarian carcinoma tissue.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • Unpurified ab32034 showing positive staining in Stomach adenocarcinoma tissue.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • Equilibrium disassociation constant (KD)
    Learn more about KD

    Click here to learn more about KD

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32034).

  • This IHC data was generated using the same anti-p27 KIP 1 antibody clone, Y236, in a different buffer formulation (cat# ab32034).

    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.

References

This product has been referenced in:
  • Singha PK  et al. TGF-ß induced TMEPAI/PMEPA1 inhibits canonical Smad signaling through R-Smad sequestration and promotes non-canonical PI3K/Akt signaling by reducing PTEN in triple negative breast cancer. Genes Cancer 5:320-36 (2014). Read more (PubMed: 25352949) »
  • Abu N  et al. Flavokawain A induces apoptosis in MCF-7 and MDA-MB231 and inhibits the metastatic process in vitro. PLoS One 9:e105244 (2014). WB ; Human . Read more (PubMed: 25286005) »
See all 10 Publications for this product

Customer reviews and Q&As

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Permeabilization
Yes - 0.5% Triton X-100
Specification
HeLa
Fixative
Paraformaldehyde

Dr. Kirk Mcmanus

Verified customer

Submitted Jan 14 2019

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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